EP2319318A2 - Verfahren zur Identifizierung von Verbindungen mit TRNA-Splicing Endonuklease als Aufhänger, und Verwendungen für diese Verbindungen als anti-Proliferationsmittel - Google Patents

Verfahren zur Identifizierung von Verbindungen mit TRNA-Splicing Endonuklease als Aufhänger, und Verwendungen für diese Verbindungen als anti-Proliferationsmittel Download PDF

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EP2319318A2
EP2319318A2 EP10184004A EP10184004A EP2319318A2 EP 2319318 A2 EP2319318 A2 EP 2319318A2 EP 10184004 A EP10184004 A EP 10184004A EP 10184004 A EP10184004 A EP 10184004A EP 2319318 A2 EP2319318 A2 EP 2319318A2
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Prior art keywords
compound
trna splicing
splicing endonuclease
cell
substrate
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French (fr)
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EP2319318A3 (de
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Christopher R. Trotta
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PTC Therapeutics Inc
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PTC Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to a method for screening and identifying compounds that modulate the activity of an animialia tRNA splicing endonuclease.
  • the invention provides assays for the identification of compounds that inhibit or reduce the activity of an animalia tRNA splicing endonuclease.
  • the methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads useful for preventing, treating, managing or ameliorating cancer or one or more symptoms thereof.
  • Cancer is the second leading cause of death in the United States.
  • the American Cancer Society estimated that in 2001, there would be 1.3 million new cases of cancer and that cancer will cause 550,000 deaths. Overall rates have declined by 1% per year during the 1990s. There are 9 million Americans alive who have ever had cancer. NIH estimates the direct medical costs of cancer as $60 billion.
  • chemotherapeutic agents there are a variety of chemotherapeutic agents available for treatment of neoplastic disease.
  • traditional chemotherapy has many drawbacks (see, for example, Stockdale, 1998, "Principles Of Cancer Patient Management” in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10 ).
  • chemotherapeutic agents are toxic, and chemotherapy can cause significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc.
  • tumor cells are resistant or develop resistance to chemotherapeutic agents through multi-drug resistance.
  • tRNAs contain a number of global as well as, species-specific modifications ( Bjork, G. Biosynthesis and Function of Modified Nucleosides, in tRNA: Structure, Biosynthesis and Function, D. Soll and U. RajBhandary (eds.), American Society for Microbiology, Washington DC, (1995), pp. 165-205 ).
  • isoaccepting groups of tRNA contain intervening sequences ranging in size from 14-105 nucleotides ( Trotta, C.R. and Abelson, J.N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction?
  • RNA World II Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999 ) and Abelson et al., 1998, Journal of Biological Chemistry 273:12685-12688 ). Removal of the intron requires the activity of 3 enzymes.
  • the tRNA is recognized and cleaved at the 5' and 3' junction by the tRNA splicing endonuclease.
  • the archaeal and eucaryal tRNA endonuclease are evolutionary conserved enzymes and contain a similar active site to achieve cleavage at the 5' and 3' splice sites. However, they have diverged to recognize the tRNA substrate in a different manner.
  • the archaeal enzyme recognizes a conserved intronic structure known as the bulge-helix-bulge. This structure is comprised of two 3-nucleotide bulges separated by a 4-nucleotide helix. Cleavage occurs within each bulge to release the intron.
  • the eucaryal endonuclease recognizes the tRNA substrate in a mature domain dependent fashion, measuring a set distance from the mature domain to the 5' and 3' splice sites ( Reyes et al., 1988, Cell 55:719-730 ).
  • the eucaryal enzyme requires a bulge at each splice site and that the enzyme has actually retained the ability to recognize tRNA by an intron-dependent recognition mechanism identical to that of the archaeal endonuclease ( Fruscoloni et al., 2001, EMBO Rep 2:217-221 ).
  • the tRNA half molecules are ligated by the action of a unique tRNA splicing ligase ( Trotta, C.R. and Abelson, J.N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction?
  • RNA World II In RNA World II, Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999 ) and Abelson et al., 1998, Journal of Biological Chemistry 273:12685-12688 ).
  • yeast the product of ligation is a tRNA with a phosphate at the splice junction. Removal of the phosphate is carried out by a tRNA 2'-phosphotransferase to yield a mature tRNA product ( Trotta, C.R. and Abelson, J.N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction? In RNA World II, Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999 ) and Abelson et al., 1998, Journal of Biological Chemistry 273:12685-12688 ).
  • tRNA is an important component in the translational machinery and is quite stable compared to various other protein-based components (elongation factors, amino-acyl synthetases, etc.). tRNA molecules have very long half-lives. Furthermore, like rRNA and ribosomes, tRNA is present in excess within the cytoplasm of actively growing cells ( Ikemura, T. and Okeki, H., 1983, Cold Spring Harbor Symp. Quant. Biol. 47:1087-1097 ). Thus, specific targeting of tRNA molecules allows a selective inhibition of uncontrolled cell proliferation and not cell growth.
  • the present invention provides methods for identifying a compound that modulates the activity of an animalia tRNA splicing endonuclease.
  • the invention provides methods for identifying a compound that inhibits the activity of an animalia tRNA splicing endonuclease.
  • the invention encompasses the use of the compounds identified utilizing the methods of the invention for the prevention, treatment, management or amelioration of a proliferative disorder or a symptom thereof.
  • the invention provides cell-based and cell-free assays for the identification of a compound that modulates the activity of an animalia tRNA splicing endonuclease, preferably a mammalian tRNA splicing endonuclease and most preferably a human tRNA splicing endonuclease.
  • These assays may be reporter gene-based assays, fluorescence resonance energy transfer (“FRET")-based assays, or fluorescence polarization assays and may be conducted in a high throughput screen format. Further, these assays directly or indirectly measure the ability of a compound to modulate an animalia tRNA splicing endonuclease.
  • FRET fluorescence resonance energy transfer
  • the ability of a compound to modulate animalia tRNA splicing endonuclease activity that was identified utilizing an indirect assay e.g ., a cell-based assay such as a reporter gene cell-based assay or a FRET cell-based assay
  • an indirect assay e.g ., a cell-based assay such as a reporter gene cell-based assay or a FRET cell-based assay
  • a more direct assay e.g., a FISH assay
  • the reporter gene-based assays may be conducted by contacting a compound with an animalia cell genetically engineered to express a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron, and measuring the expression of said reporter gene.
  • the reporter gene-based assays may be conducted by contacting a compound with an animalia cell-free extract and a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron, and measuring the expression of said reporter gene.
  • reporter gene expression indicates that a particular compound modulates the activity of the tRNA splicing endonuclease.
  • a decrease in reporter gene expression relative to a previously determined reference range, or to the expression in the absence of the compound or the presence of an appropriate control (e.g ., a negative control) in such reporter-gene based assays indicates that a particular compound reduces or inhibits the activity of an animalia tRNA splicing endonuclease ( e.g ., the recognition or cleavage of a tRNA intron).
  • an appropriate control e.g a negative control
  • reporter gene expression indicates that a particular compound enhances the activity of an animalia tRNA splicing endonuclease.
  • the invention provides a method for identifying a compound that modulates animalia tRNA splicing endonuclease activity, said method comprising: (a) expressing a nucleic acid comprising a reporter gene in a cell, wherein the reporter gene comprises a tRNA intron; (b) contacting said cell with a member of a library of compounds; and (c) detecting the expression of said reporter gene, wherein a compound that modulates tRNA splicing endonuclease activity is identified if the expression of said reporter gene in the presence of a compound is altered relative to a previously determined reference range, or the expression of said reporter gene in the absence of the compound or the presence of an appropriate control ( e . g .,. a negative control).
  • an appropriate control e . g .,. a negative control
  • the invention provides a method for identifying a compound that modulates animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting a member of a library of compounds with a cell containing a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron; and (b) detecting the expression of said reporter gene, wherein a compound that modulates tRNA splicing endonuclease activity is identified if the expression of said reporter gene in the presence of a compound is altered relative to a previously determined reference range, or the expression of said reporter gene in the absence of said compound or the presence of an appropriate control (e.g ., a negative control).
  • an appropriate control e.g ., a negative control
  • the invention provides a method for identifying a compound that modulates animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting a member of a library of compounds with a cell-free extract and a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron; and (b) detecting the expression of said reporter gene, wherein a compound that modulates tRNA splicing endonuclease activity is identified if the expression of said reporter gene in the presence of a compound is altered relative to a previously determined reference range, or the expression of said reporter gene in the absence of said compound or the presence of an appropriate control (e.g ., a negative control).
  • an appropriate control e.g ., a negative control
  • the step of contacting a compound with a cell, or cell-free extract and a nucleic acid in the reporter gene-based assays described herein is preferably conducted in an aqueous solution comprising a buffer and a combination of salts (such as KCl, NaCl and/or MgCl 2 ).
  • aqueous solution comprising a buffer and a combination of salts (such as KCl, NaCl and/or MgCl 2 ).
  • the optimal concentration of each salt used in the aqueous solution is dependent on the endonuclease and the compounds used, and can be determined using routine experimentation.
  • the aqueous solution approximates or mimics physiologic conditions.
  • the aqueous solution further comprises a detergent or a surfactant.
  • the reporter gene constructs utilized in the reporter gene-based assays described herein may comprise the coding region of a reporter gene and a tRNA intron that renders the mRNA coding the reporter gene out of frame.
  • the reporter gene constructs utilized in the reporter gene-based assays described herein comprise a tRNA intron within the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated regions.
  • the tRNA intron interrupts an mRNA splicing element.
  • a reporter gene construct utilized in the reporter gene-based assays described herein comprises the coding region of a reporter gene and a tRNA intron within the open reading frame of the reporter gene.
  • the intron utilized in the reporter gene constructs described herein may comprise a bulge-helix-bulge conformation.
  • a reporter gene construct utilized in the reporter-gene-based assays described herein comprises a mature domain containing a tRNA intron.
  • reporter gene well-known to one of skill in the art may be utilized in the reporter gene constructs described herein.
  • reporter genes include, but are not limited to, the gene encoding firefly luciferase, the gene coding renilla luciferase, the gene encoding click beetle luciferase, the gene encoding green fluorescent protein, the gene encoding yellow fluorescent protein, the gene encoding red fluorescent protein, the gene encoding cyan fluorescent protein, the gene encoding blue fluorescent protein, the gene encoding beta-galactosidase, the gene encoding beta-glucoronidase, the gene encoding beta-lactamase, the gene encoding chloramphenicol acetyltransferase, and the gene encoding alkaline phosphatase.
  • the reporter gene-based assays described herein maybe conducted in a cell genetically engineered to express a reporter gene or in vitro utilizing a cell-free extract.
  • a cell-free extract may be derived from any cell or cell line of any species well-known to one of skill in the art. Examples of cells and cell types include, but are not limited to, human cells, cultured mouse cells, cultured rat cells or Chinese hamster ovary ("CHO") cells.
  • Fluoroscent resonance energy transfer (“FRET") assays may be used to identify a compound that modulates the activity of an animalia tRNA splicing endonuclease.
  • the FRET assays may be conducted utilizing labeled subunits of an animalia tRNA splicing endonuclease or labeled substrates for an animalia tRNA splicing endonuclease.
  • the FRET cell-based assays may be conducted by microinjecting or transfecting a substrate for an animalia tRNA splicing endonuclease into an animalia cell and contacting the cell with a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore, and measuring the fluorescence of the substrate by, e.g ., fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, inhibit or reduce the production of a detectable fluorescent signal.
  • a compound that enhances the activity of the endogenous endonuclease will enhance the cleavage of the substrate and thus, increase the production of a detectable fluoroscent signal.
  • the FRET cell-based assays may be conducted by microinjecting or transfecting a substrate for an animalia tRNA splicing endonuclease into a cell and contacting the cell with a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluoroscent donor moiety, and measuring the fluorescence of the substrate by, e . g ., fluoresence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • fluoresence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in a decrease in the fluorescence emission by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce cleavage of the substrate and thus, increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that enhances the activity of the endogenous tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • an agent known to inhibit or reduce the activity of an animalia tRNA splicing ligase such as an antibody that specifically binds to an animalia tRNA splicing ligase is included in the contacting step of the FRET assays to exclude the possibility that the compound is solely inhibiting or reducing the activity of the ligase.
  • an animalia cell or an animalia cell-free extract that is deficient in tRNA splicing ligase is used in the FRET assays.
  • ATP may be excluded from the assay.
  • any effect of a compound in the FRET assay in the absence of ATP cannot be attributed to an effect on the ligase reaction and is therefore an effect on an animalia tRNA splicing endonuclease.
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) microinjecting or transfecting a substrate of a tRNA splicing endonuclease into an animalia cell, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher and labeled with a fluorophore; (b) contacting the cell with a member of a library of compounds; and (c) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if a fluorescent signal is not detectable in the presence of the compound relative to the absence of the compound or the presence of
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell containing a substrate of a tRNA splicing endonuclease with a member of a library of compounds, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher or labeled at the 3' end with a fluorophore; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if a fluorescent signal is not detectable in the presence of the compound relative to the absence of the compound or the presence of a negative control.
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) microinjecting or transfecting a substrate of a tRNA splicing endonuclease into a animalia cell, wherein said substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled with at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety; (b) contacting the cell with a member of a library of compounds; and (c) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell containing substrate of a tRNA splicing endonuclease with a member of a library of compounds, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if the fluorescence emission of the fluorescent acceptor moiety at the wavelength of a fluorescent donor moiety in the presence of the compound is decreased relative to the absence
  • the FRET cell-free-based assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease with an animalia cell-free extract (preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore, and measuring the fluorescence of the substrate by, e.g ., a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease in the animalia cell-free extract or the purified animalia tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits the activity of the animalia tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, inhibit or reduce the production of a detectable fluorescent signal.
  • a compound that enhances the activity of the animalia tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, increase the production of a detectable fluorescent signal.
  • the FRET cell-free-based assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease with an animalia cell-free extract or a purified animalia tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g ., a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease in the animalia cell-free extract or the purified animalia tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits the activity of the tRNA splicing endonuclease will inhibit or reduce cleavage of the substrate and thus, increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that enhances the activity of the endogenous tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell-free extract (preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease with a substrate of a tRNA splicing endonuclease and a member of a library of compounds, wherein the substrate is labeled at the 5' end with a fluorophore and at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell-free extract (preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease with a substrate of a tRNA splicing endonuclease and a member of a library of compounds, wherein said substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduce
  • the substrates for a tRNA splicing endonuclease utilized in the FRET assays described herein comprise an intron.
  • the substrate for a tRNA splicing endonuclease utilized in the FRET assays described herein comprises a tRNA intron.
  • the intron may have a bulge-helix-bulge conformation
  • the substrate comprises a mature domain that contains an intron.
  • the effect of a compound on the activity of an animalia tRNA splicing endonuclease may be determined utilizing a fluorescence polarization-based assay.
  • a fluorescently labeled substrate for an animalia tRNA splicing endonuclease is contacted with an animalia cell-free extract or a purified animalia tRNA splicing endonuclease and a compound or member of a library of compounds; and the fluorescent polarized light emitted is measured utilizing techniques well-known to one of skill in the art or described herein, wherein an alteration in the fluorescently polarized light emitted relative to a control or the absence of the compound or the member of a library of compounds indicates that the compound or member of a library of compounds modulates animalia tRNA splicing endonuclease activity.
  • a host cell is engineered to contain a reporter gene and a suppressor tRNA, wherein the reporter gene construct comprises a reporter gene with a nonsense codon in its open reading frame such that the open reading frame is interrupted and the suppressor tRNA's expression is regulated by an inducible regulatory element and the suppressor tRNA contains a tRNA intron in the antisense codon; the expression of the suppressor tRNA is induced; the host cell is contacted with a compound; and the expression of the reporter gene and/or the activity of the protein encoded by the reporter gene is measured utilizing techniques well-known to one of skill in the art or described herein.
  • a compound that inhibits or reduces the activity of an animalia tRNA splicing endonuclease will inhibit or reduce the production of functional suppressor tRNA and thus, reduce the expression of the reporter gene relative to a previously determined reference range, or the expression of the reporter gene in the absence of the compound or the presence of an appropriate control (e.g ., a negative control).
  • a compound that enhances the activity of an animalia tRNA splicing endonuclease will enhance the production of functional suppressor tRNA and thus, enhance the production of the reporter gene relative to a previously determined reference range, or the expression of the reporter gene in the absence of the compound or the presence of an appropriate control ( e.g ., a negative control).
  • the assays of the present invention can be performed using different incubation times.
  • the cell-free extract or the purified tRNA splicing endonuclease and substrate for animalia tRNA splicing endonuclease can be incubated together before the addition of a compound or a member of a library of compounds.
  • the cell-free extract or the purified animalia tRNA splicing endonuclease are incubated with a substrate for animalia tRNA splicing endonuclease before the addition of a compound or a member of a library of compounds for at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • cell-free extract or purified animalia tRNA splicing endonuclease, or a substrate for animalia tRNA splicing endonuclease is incubated with a compound or a member of a library of compounds before the addition of the substrate, or the cell-free extract or the purified animalia tRNA splicing endonuclease, respectively.
  • a compound or a member of a library of compounds is incubated with a substrate for animalia tRNA splicing endonuclease or cell-free extract or purified animalia tRNA splicing endonuclease before the addition of the remaining component, i.e ., cell-free extract or purified animalia tRNA splicing endonuclease, or substrate for animalia tRNA splicing endonuclease, respectively, is at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • the reaction vessel comprises the three components, i.e ., a compound or a member of a library of compounds, the cell-free extract or the purified animalia tRNA splicing endonuclease, and substrate for animalia tRNA splicing endonuclease
  • the reaction may be further incubated for at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • the progress of the reaction can be measured continuously. For example, if a substrate for animalia tRNA splicing endonuclease or subunits of animalia tRNA splicing endonuclease are labeled with fluorophore(s), the progress of the reaction can be monitored continuously using a fluorescence emission detector such as a Viewlux or Analyst. Alternatively, time-points may be taken at different times of the reaction to monitor the progress of the reaction.
  • Certain assays of the present invention are indirect assays for compounds that affect animalia tRNA splicing endonuclease and may detect compounds that affect another aspect of the tRNA splicing pathway.
  • any assay that measures the direct effect of the compound on animalia tRNA splicing endonuclease activity can be performed.
  • Such assays include assays using purified animalia tRNA splicing endonuclease and are described below.
  • the compounds utilized in the assays described herein may be members of a library of compounds.
  • the compound is selected from a combinatorial library of compounds comprising peptoids; random biooligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and small organic molecule libraries.
  • the small organic molecule libraries are libraries of benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, or diazepindiones.
  • the compounds are screened in pools. Once a positive pool has been identified, the individual compounds of that pool are tested separately.
  • the pool size is at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, or at least 500 compounds.
  • the structure of the compound may be determined utilizing well-known techniques or by referring to a predetermined code.
  • the structure of the compound may be determined by mass spectroscopy, NMR, vibrational spectroscopy, or X-ray crystallography.
  • a compound identified in accordance with the methods of the invention may disrupt the interaction of the subunits of an animalia tRNA splicing endonuclease.
  • a compound identified in accordance with the methods of the invention may insert itself into teh active site of an animalia tRNA splicing endonuclease.
  • a compound identified in accordance with the methods of the invention may directly bind to the tRNA splicing endonuclease.
  • a compound identified in accordance with the methods of invention may bind to the intron.
  • a compound identified in accordance with the methods of invention may also disrupt an interaction between a tRNA intron and a tRNA splicing endonuclease.
  • a compound identified in accordance with the methods of the invention may disrupt the interaction between the tRNA mature domain and the tRNA splicing endonuclease.
  • a compound identified in accordance with the methods of the invention inhibits animalia tRNA splicing endonuclease activity.
  • a compound identified in accordance with the methods of the invention inhibits preferentially inhibits animalia tRNA splicing endonuclease activity.
  • the compound identified using the assays described herein is a small molecule.
  • the compound identified using the assays described herein is not known to affect the activity of an animalia tRNA splicing endonuclease.
  • the compound identified using the assays described herein has not been used as or suggested to be an anti-proliferative agent or an antifungal agent.
  • a compound that modulates the activity of a tRNA splicing endonuclease described herein may be tested in in vitro assays or in vivo assays (e.g ., cell-based assays or cell-free assays) well-known to one of skill in the art or described herein for the effect of said compound on mRNA translation.
  • the compounds identified by the methods of the present invention can be screened for their effect on the production of mature tRNA from any of the 28 intron containing human pre-tRNAs.
  • In vitro and in vivo assays well-known to one of skill in the art or described herein may be used to determine the antiproliferative effect of a particular compound on hyperproliferative cells versus normal cells.
  • a particular compound identified utilizing the assays described herein may be tested in an animal model for cancer to determine the efficacy of the compound in the prevention, treatment or amelioration of cancer or a symptom thereof.
  • the effect of a compound identified utilizing the assays described herein may be tested for its effect on yeast tRNA splicing endonculease.
  • the invention provides for methods for preventing, treating, managing or ameliorating a proliferative disorder or a symptom thereof, said method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a compound, or a pharmaceutically acceptable salt thereof, identified according to the methods described herein.
  • the invention provides for a method of preventing, treating, managing or ameliorating cancer or a symptom thereof, said method comprising administering to a subject in need thereof an effective amount of a compound, or a pharmaceutically acceptable salt thereof, identified according to the methods described herein.
  • the invention provides a method of identifying a therapeutic agent for the prevention, treatment, management or ameliorating of cancer or a symptom thereof, said method comprising: (a) contacting a member of a library of compounds with a cell containing a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron; and (b) detecting the expression of said reporter gene, wherein if a compound that reduces the expression of said reporter gene relative to a previously determined reference range or the expression of said reporter gene in the absence of said compound or the presence of an appropriate control (e.g ., a negative control such as PBS) is detected in (b), then (c) contacting the compound with a cancer cell or a neoplastic cell and detecting the proliferation of said cancer cell or neoplastic cell, so that if the compound reduces or inhibits the proliferation of the cancer cell or neoplastic cell, the compound is identified as an antiproliferative compound.
  • the compound e.g ., a negative
  • tRNA splicing endonuclease should only be toxic to highly proliferative transformed, malignant cells, while allowing for normal cellular growth and metabolism because not all tRNAs require splicing and tRNA splicing occurs more frequently in proliferating cells.
  • tRNA species that require removal of intronic sequences ( Trotta, C.R. and Abelson, J.N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction? In RNA World II, Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999 )).
  • the current version of the sequence of the human genome has identified 648 tRNA species.
  • tRNA splicing endonuclease By targeting the tRNA splicing endonuclease, an enzyme dedicated to removal of tRNA introns, the inhibition of tRNA production is fine-tuned to a very few essential tRNA molecules (potentially only a single tRNA). Thus, by inhibiting this process, a very mild toxicity, if any, to normal cells will be produced, while the ability of rapidly proliferating transformed cells to divide will be reduced or ablated as a result of the loss in translational capacity.
  • the invention further provides methods for verifying or confirming the ability of a compound to modulate the activity of a tRNA splicing endonuclease.
  • the ability of a compound to modulate the activity of a tRNA splicing endonuclease can be verified or confirmed utilizing any of the assays described herein to identify such a compound.
  • the invention provides a method for verifying the ability of a compound to inhibit animalia tRNA splicing endonuclease activity, said method comprising: (a) expressing a nucleic acid comprising a reporter gene in a cell, wherein the reporter gene comprises a tRNA intron; (b) contacting said cell with a compound; and (c) detecting the expression of said reporter gene, wherein a compound that inhibits tRNA splicing endonuclease activity is verified if the expression of said reporter gene in the presence of a compound is reduced as compared to the expression of said reporter gene in the absence of said compound or the presence of a control.
  • the invention provides a method for verifying the ability of a compound to inhibit animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting a compound with a cell-free extract and a nucleic acid comprising a reporter gene, wherein the reporter gene comprises an intron; and (b) detecting the expression of said reporter gene, wherein a compound that inhibits tRNA splicing endonuclease activity is verified if the expression of said reporter gene in the presence of a compound is reduced as compared to the expression of said reporter gene in the absence of said compound or the presence of a control.
  • the invention provides a method for verifying the ability of a compound to inhibit animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting a member of a library of compounds with a cell containing a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron; and (b) detecting the expression of said reporter gene, wherein a compound that inhibits tRNA splicing endonuclease activity is verified if the expression of said reporter gene in the presence of a compound is reduced as compared to the expression of said reporter gene in the absence of said compound or the presence of a control.
  • the term "compound” refers to any agent or complex that is being tested for its ability to modulate tRNA splicing endonuclease or has been identified as modulating tRNA splicing endonuclease activity.
  • disorder and “disease” are to refer to a condition in a subject.
  • the term "effective amount” refers to the amount of a compound which is sufficient to reduce or ameliorate the progression, severity and/or duration of a proliferative disorder or one or more symptoms thereof, prevent the development, recurrence or onset of a proliferative disorder or one or more symptoms thereof, prevent the advancement of a proliferative disorder or one or more symptoms thereof, or enhance or improve the therapeutic(s) effect(s) of another therapy.
  • fluorescent acceptor moiety refers to a fluorescent compound that absorbs energy from a fluorescent donor moiety and re-emits the transferred energy as fluorescence.
  • fluorescent acceptor moieties include, but are not limited to, coumarins and related fluorophores, xanthenes ( e.g ., fluoresceins, rhodols, and rhodamines), resorufins, cyanines, difluoroboradiazindacenes and phthalocyanines.
  • fluorescent donor moiety refers to a fluorescent compound that can absorb energy and is capable of transferring the energy to an acceptor, such as another fluorescent compound.
  • fluorescent donor moieties include, but are not limited to, coumarins and related dyes, xanthene dyes ( e.g ., fluoresceins, rhodols and rhodamines), resorufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides ( e .
  • fluorophore refers to a chromophore that fluoresces.
  • the term "host cell” refers includes a particular subj ect cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in: succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • the term "in combination” refers to the use of more than one therapy (e.g ., prophylactic and/or therapeutic agents).
  • the use of the term “in combination” does not restrict the order in which therapies (e . g ., prophylactic and/or therapeutic agents) are administered to a subject with a proliferative disorder.
  • a first therapy e.g ., a prophylactic or therapeutic agent such as a compound identified in accordance with the methods of the invention
  • a prophylactic or therapeutic agent such as a compound identified in accordance with the methods of the invention
  • can be administered prior to e.g ., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before
  • a second therapy e.g ., a prophylactic or therapeutic agent such as a chemotherapeutic agent or a TNF- ⁇ antagonist
  • a library refers to a plurality of compounds.
  • a library can be a combinatorial library, e.g ., a collection of compounds synthesized using combinatorial chemistry techniques, or a collection of unique chemicals of low molecular weight (less than 1000 daltons) that each occupy a unique three-dimensional space.
  • ORF refers to the open reading frame of a mRNA, i . e ., the region of the mRNA that is translated into protein.
  • the terms “manage”, “managing” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g ., a prophylactic or therapeutic agent) which does not result in a cure of the proliferative disorder.
  • a subject is administered one or more therapies to "manage” a disease or disorder so as to prevent the progression or worsening of the disease or disorder.
  • non-responsive and refractory describe patients treated with a currently available therapy (e.g ., prophylactic or therapeutic agent) for a proliferative disorder (e . g ., cancer), which is not clinically adequate to relieve one or more symptoms associated with such proliferative disorder.
  • a currently available therapy e.g ., prophylactic or therapeutic agent
  • a proliferative disorder e.g ., cancer
  • Such patients suffer from severe, persistently active disease and require additional therapy to ameliorate the symptoms associated with their proliferative disoder.
  • salts include, but is not limited to, salts of acidic or basic groups that may be present in compounds identified using the methods of the present invention.
  • Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • the acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tarmate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate
  • 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts e ., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts.
  • Compounds that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
  • Compounds that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, and iron salts.
  • the terms "prevent”, “preventing” and “prevention” refer to the prevention of the development, recurrence or onset of a proliferative disorder or one or more symptoms thereof resulting from the administration of one or more compounds identified in accordance the methods of the invention or the administration of a combination of such a compound and a known therapy for a proliferative disorder.
  • the term "previously determined reference range” refers to a reference range for the readout of a particular assay. In a specific embodiment, the term refers to a reference range for the expression and/or the activity of a reporter gene by a particular cell or in a particular cell-free extract. Each laboratory will establish its own reference range for each particular assays, each cell type and each cell-free extract. In a preferred embodiment, at least one positive control and at least one negative control are included in each batch of compounds analyzed.
  • the terms “prophylactic agent” and “prophylactic agents” refer to any agent(s) which can be used in the prevention of a proliferative disorder.
  • the term “prophylactic agent” refers to a compound identified in the screening assays described herein.
  • the term “prophylactic agent” refers to an agent other than a compound identified in the screening assays described herein which is known to be useful for, or has been or is currently being used to prevent or impede the onset, development and/or progression of a proliferative disorder or one or more symptoms thereof.
  • prophylactically effective amount refers to the amount of a therapy (e.g ., a prophylactic agent) which is sufficient to result in the prevention of the development, recurrence or onset of one or more symptoms associated with a proliferative disorder.
  • the term "purified,” in the context of a compound, e.g ., a compound identified in accordance with the method of the invention, refers to a compound that is substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the compound is 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, or 99% free of other, different compounds.
  • a compound identified in accordance with the methods of the invention is purified.
  • the term "purified,” in the context of a proteinaceous agent refers to a proteinaceous agent which is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a proteinaceous agent in which the proteinaceous agent is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a proteinaceous agent that is substantially free of cellular material includes preparations of a proteinaceous agent having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein, polypeptide, peptide, or antibody (also referred to as a "contaminating protein").
  • the proteinaceous agent is recombinantly produced, it is also preferably substantially free of culture medium, i.e ., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the proteinaceous agent is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e ., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the proteinaceous agent. Accordingly, such preparations of a proteinaceous agent have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the proteinaceous agent of interest
  • quencher refers to a molecule or a part of a compound that is capable of reducing the emission from a fluorescent moiety. Such reduction includes reducing the light after the time when a photon is normally emitted from a fluorescent moiety.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds ( i.e ,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, organic or inorganic compounds having a molecular weight less than about 100 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Salts, esters, and other pharmaceutically acceptable forms of such compounds are also encompassed.
  • the terms “subject” and “patient” are used interchangeably herein.
  • the terms “subject” and “subjects” refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g ., a chimpanzee, a monkey such as a cynomolgous monkey and a human), and more preferably a human.
  • the subject is refractory or non-responsive to current therapies for a proliferative disorder.
  • the subject is a farm animal (e.g ., a horse, a cow, a pig, etc.) or a pet ( e.g ., a dog or a cat).
  • the subject is a human.
  • a substrate for an animalia tRNA splicing endonuclease refers to any nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease.
  • a nucleotide sequence comprising a bulge-helix-bulge structure or a mature domain of a precursor tRNA may be utilized as a substrate for an animalia tRNA splicing endonuclease in an assay described herein.
  • a nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease may comprise 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, or more.
  • the substrates for a tRNA splicing endonuclease utilized in the assays described herein comprise a tRNA intron.
  • the substrate may comprise a mature domain or a bulge-helix-bulge conformation.
  • the substrate comprises a mature domain of a precursor tRNA.
  • a substrate for an animalia tRNA endonuclease may be produced by any method well-known to one of skill in the art.
  • the substrate may be chemically synthesized using phosphoramidite or other solution or solid-phase methods.
  • phosphoramidite or other solution or solid-phase methods.
  • Detailed descriptions of the chemistry used to form polynucleotides by the phosphoramidite method are well known (see, e.g., Caruthers et al., U.S. Pat. Nos. 4,458,066 and 4,415,732 ; Caruthers et al., 1982, Genetic Engineering 4:1-17 ; Users Manual Model 392 and 394 Polynucleotide Synthesizers, 1990, pages 6-1 through 6-22, Applied Biosystems, Part No.
  • the substrate can be purified using standard techniques known to those skilled in the art (see Hwang et al., 1999, Proc. Natl. Acad. Sci. USA 96(23):12997-13002 and references cited therein).
  • purification techniques include, but are not limited to, reverse-phase high-performance liquid chromatography ("reverse-phase HPLC”), fast performance liquid chromatography (“FPLC”), and gel purification.
  • the substrates depicted in Figure 1 are utilized in the assays described herein.
  • both strands of the hybridized substrate are transcribed separately and the two strands are subsequently hybridized by heating and cooling.
  • the RNA is transcribed from the 5' end in the intron (see Figure 1C ) to the 3' end in the intron.
  • the term "synergistic” refers to a combination of a compound identified using one of the methods described herein, and another therapy (e.g ., agent) which has been or is currently being used to prevent, treat, manage or ameliorate a proliferative disorder or a symptom thereof, which is more effective than the additive effects of the therapies.
  • a synergistic effect of a combination of therapies permits the use of lower dosages of one or more of the therapies and/or less frequent administration of said therapies to a subj ect with a proliferative disorder.
  • a therapy e.g ., a prophylactic or therapeutic agent
  • a synergistic effect can result in improved efficacy of therapies (e.g ., agents) in the prevention, treatment, management or amelioration of a proliferative disorder.
  • a synergistic effect of a combination of therapies e.g ., prophylactic or therapeutic agents may avoid or reduce adverse or unwanted side effects associated with the use of either therapy alone.
  • the terms “therapeutic agent” and “therapeutic agents” refer to any agent(s) which can be used in the prevention, treatment, management or amelioration of one or more symptoms of a proliferative disorder.
  • the term “therapeutic agent” refers to a compound identified in the screening assays described herein.
  • the term “therapeutic agent” refers to an agent other than a compound identified in the screening assays described herein which is known to be useful for, or has been or is currently being used to prevent, treat, manage or ameliorate a proliferative disorder or one or more symptoms thereof.
  • the term "therapeutically effective amount” refers to that amount of a therapy (e.g ., a therapeutic agent) sufficient to result in the amelioration of one or more symptoms of a proliferative disorder, prevent advancement of a proliferative disorder, cause regression of the proliferative disorder, or to enhance or improve the therapeutic effect(s) of another therapy (e.g ., therapeutic agent).
  • a therapy e.g ., a therapeutic agent
  • a therapeutically effective amount refers to the amount of a therapy (e.g ., a therapeutic agent) that inhibits or reduces the proliferation of cancerous cells, inhibits or reduces the spread of tumor cells (metastasis), inhibits or reduces the onset, development or progression of one or more symptoms associated with cancer, or reduces the size of a tumor.
  • a therapy e.g ., a therapeutic agent
  • a therapeutically effective of a therapy reduces the proliferation of cancerous cells or the size of a tumor by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control such as phosphate buffered saline ("PBS").
  • PBS phosphate buffered saline
  • the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of a proliferative disorder or one or more symptoms thereof resulting from the administration of one or more compounds identified in accordance the methods of the invention, or a combination of one or more compounds identified in accordance with the invention and another therapy.
  • such terms refer to the inhibition or reduction in the proliferation of cancerous cells, the inhibition or reduction the spread of tumor cells (metastasis), the inhibition or reduction in the onset, development or progression of one or more symptoms associated with cancer, or the reduction in the size of a tumor.
  • tRNA intron refers to any nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease.
  • tRNA intron refers to an intron typically found in a precursor tRNA.
  • tRNA splicing endonuclease refers to the enzyme that is responsible for the recognition of the splice sites contained in precursor tRNA and the cleavage of the introns present in precursor tRNA.
  • the archaeal tRNA splicing endonuclease recognizes the bulge-helix-bulge motif in archaeal precursor tRNA.
  • the eukaryotic tRNA splicing endonuclease recognizes the splice sites contained in precursor tRNA by measuring the distance from the mature domain to the splice sites.
  • the eukaryotic tRNA splicing endonuclease also has the capacity to recognize a bulge-helix-bulge motif contained in precursor tRNA.
  • the yeast tRNA endonuclease is a heterotetramer comprising subunits having the molecular masses of 54 kDa (SEN54), 44 kDa (SEN2), 34 kDa (SEN 34), and 15 kDa (SEN 15).
  • the human homologs of the SEN2 and SEN34 subunits have been identified and the amino acid sequences can be found in GenBank under accession numbers NP_079541 and XP_085899, respectively.
  • the tRNA splicing endonuclease utilized in the assays described herein is derived from or encodes an animal tRNA splicing endonuclease (preferably, a mammalian tRNA splicing endonuclease).
  • the tRNA splicing endonuclease utilized in the assays described herein is a human tRNA splicing endonuclease.
  • the terms “therapy” and “therapies” refer to any method, protocol and/or agent that can be used in the prevention, treatment, management or amelioration of a disesase or disorder (e.g ., a proliferative disorder or a fungal infection) or one or more symptoms thereof.
  • a disesase or disorder e.g ., a proliferative disorder or a fungal infection
  • such terms refer to chemotherapy, radiation therapy, surgery, supportive therapy and/or other therapies useful in the prevention, treatment, management or amelioration of a disease or disorder (e.g ., a proliferative disorder or a fungal infection) or one or more symptoms thereof known to skilled medical personnel.
  • tRNA splicing endonuclease extract refers to an extract from a cell containing tRNA splicing endonuclease activity.
  • a tRNA splicing endonuclease extract is a cell-extract containing tRNA splicing endonuclease activity and the components necessary for the transcription and translation of a gene.
  • Figure 1 Substrates for HTS Fluorescent screening.
  • the endogenous tRNA is shown in panel A; the hybridized tRNA substrate is shown in panel B; and the circularly permuted tRNA substrate is shown in panel C.
  • the 5' ss designates the 5' splice site and 3' ss designates the 3' splice site.
  • Figure 2 Amino Acid Sequence Alignment of human (Hs Sen 2 (SEQ ID NO: 1) and Hs Sen 2 var. (SEQ ID NO: 2)) and yeast (Sc Sen 2p (SEQ ID NO: 3)) tRNA splicing endonuclease Sen 2 submit.
  • the boxed amino acid residues indicate the YRGGY (SEQ ID NO: 4) active site motif
  • the circled amino acid residue indicates the active site histidine
  • the underlined amino acid residues indicate the yeast putative transmembrane domain.
  • the present invention provides methods for identifying compounds that modulate the activity of an animalia tRNA splicing endonuclease.
  • the invention provides simple, rapid and sensitive methods for identifying compounds that inhibit the activity of a mammalian tRNA splicing endonuclease.
  • the cell-based and cell-free assays described herein can be utilized in a high throughput format to screen libraries of compounds to identify those compounds that inhibit or reduce the activity of an animalia tRNA splicing endonuclease.
  • Reporter gene-based assays can be utilized to identify a compound that modulates the activity of an animalia tRNA splicing endonuclease.
  • the reporter gene-based assays described herein may be conducted by contacting a compound with a cell genetically engineered to express a nucleic acid comprising a reporter gene, wherein said reporter gene comprises a tRNA intron, and measuring the expression of said reporter gene.
  • the reporter gene-based assays may be conducted by contacting a compound with a cell-free extract and a nucleic acid comprising a reporter gene, wherein said reporter gene comprises a tRNA intron, and measuring the expression of said reporter gene.
  • reporter gene expression indicates that a particular compound modulates an animalia tRNA splicing endonuclease activity.
  • FRET assays can be utilized to identify a compound that modulates the activity of an animalia tRNA splicing endonuclease.
  • the FRET cell-based assays described herein may be conducted by microinjecting or transfecting ( e.g ., using liposomes or electroporation) a substrate for an animalia tRNA splicing endonuclease into a cell and contacting the cell with a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a flurophore, and measuring the fluorescence of the substrate by, e.g ., fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will prevent the production of a detectable fluorescent signal.
  • the FRET cell-based assays may be conducted by microinjecting or transfecting a substrate for an animalia tRNA splicing endonuclease into a cell and contacting the cell with a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g ., fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits the activity of the endogenous tRNA splicing endonuclease will reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • the FRET cell-free-based assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease with a cell-free extract or a purified animalia tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or, alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore, and measuring the fluorescence of the substrate by, e . g ., a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing will cleave the substrate and result in the production of a detectable fluorescent signal relative to a control.
  • a compound that enhances the activity of the tRNA splicing endonuclease will result in the increased fluorescence emission of a fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the activity of the tRNA splicing endonuclease will prevent or reduce the production of a detectable fluorescent signal relative to a control.
  • the FRET cell-free-based assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease with a cell-free extract or a purified animalia tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g ., a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the activity of the tRNA splicing endonuclease will increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that enhances the activity of the tRNA splicing endonuclease will reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound may be tested for its ability to enhance or inhibit the activity of an animalia tRNA endonuclease using a cell-free fluorescence polarization assay.
  • a substrate of the animalia tRNA endonuclease is labeled on its 5' or 3' end such that cleavage by the animalia tRNA endonuclease results in a decrease of size of the labeled portion of the substrate and thus in a change of fluoresence polarization.
  • the labeled substrate of the animalia tRNA endonuclease is incubated with a cell-free extract or a purified animalia tRNA splicing endonuclease and a compound to be tested.
  • a compound that enhances the activity of the tRNA splicing endonuclease activity will increase the rotation of the substrate relative to a negative control or the absence of the compound, which will result in more of the light emitted being depolarized.
  • a compound that reduces the activity of the tRNA splicing endonuclease activity will decrease the rotation of the substrate relative to a negative control or the absence of the compound which will result in the emitted light remaining polarized.
  • a compound may be tested for its ability to enhance or inhibit the activity of an animalia tRNA endonuclease using a tRNA endonuclease suppression assay or FISH assay.
  • the compounds identified in assays described herein that modulate animalia tRNA splicing endonuclease activity may be tested in in vitro assays (e.g ., cell-based assays or cell-free assays) or in vivo assays well-known to one of skill in the art or described herein for the effect of said compounds on tRNA processing and ultimately mRNA translation.
  • in vitro and in vivo assays well-known to one of skill in the art or described herein may be used to determine the antiproliferative effect of a particular compound on hyperproliferative cells versus normal cells.
  • a particular compound identified utilizing the assays described herein may be tested in an animal model for cancer to determine the efficacy of the compound in the prevention, treatment or amelioration of cancer or a symptom thereof
  • the effect of a compound identified utilizing the assays described herein may be tested for its effect on fungal tRNA splicing endonculease.
  • the structure of the compounds identified in the assays described herein that modulate animalia tRNA splicing endonuclease activity can be determined utilizing assays well-known to one of skill in the art or described herein. The methods used will depend, in part, on the nature of the library screened. For example, assays or microarrays of compounds, each having an address or identifier, may be deconvoluted, e.g ., by cross-referencing the positive sample to an original compound list that was applied to the individual test assays. Alternatively, the structure of the compounds identified herein may be determined using mass spectrometry, nuclear magnetic resonance ("NMR"), X ray crystallography, or vibrational spectroscopy.
  • NMR nuclear magnetic resonance
  • the invention encompasses the use of the compounds that inhibit or reduce the activity of an animalia tRNA splicing endonculease which were identified in accordance with the methods described herein for the prevention, treatment, management or amelioration of a proliferative disorder or one or more symptoms thereof.
  • the invention encompasses the use of the compounds that inhibit or reduce the activity of an animalia tRNA splicing endonculease which were identified in accordance with the methods described herein for the prevention, treatment, management or amelioration of cancer or one or more symptoms thereof.
  • the invention provides for specific vectors comprising a reporter gene comprising a tRNA intron operably linked to one or more regulatory elements and host cells transfected with the vectors.
  • the invention also provides for the in vitro translation of a reporter gene flanked by one or more regulatory elements.
  • Techniques for practicing this specific aspect of this invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and recombinant DNA manipulation and production, which are routinely practiced by one of skill in the art. See, e.g ., Sambrook, 1989, Molecular Cloning, A Laboratory Manual, Second Editi on; DNA Cloning, Volumes I and II (Glover, Ed. 1985 ); Oligonucleotide Synthesis (Gait, Ed.
  • reporter gene any reporter gene well-known to one of skill in the art may be used in reporter gene constructs to ascertain the effect of a compound on an animalia tRNA endonuclease.
  • Reporter genes refer to a nucleotide sequence encoding a protein that is readily detectable either by its presence or activity. Reporter genes may be obtained and the nucleotide sequence of the elements determined by any method well-known to one of skill in the art.
  • the nucleotide sequence of a reporter gene can be obtained, e.g ., from the literature or a database such as GenBank.
  • a polynucleotide encoding a reporter gene may be generated from nucleic acid from a suitable source.
  • a nucleic acid encoding the reporter gene may be chemically synthesized or obtained from a suitable source (e.g ., a cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the reporter gene) by PCR amplification.
  • a suitable source e.g ., a cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the reporter gene
  • nucleotide sequence of a reporter gene may be manipulated using methods well-known in the art for the manipulation of nucleotide sequences, e.g ., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • reporter genes include, but are not limited to, luciferase (e.g ., firefly luciferase, renilla luciferase, and click beetle luciferase), green fluorescent protein (“GFP”) (e.g ., green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, and blue fluorescent protein), beta-galactosidase (“beta-gal”), beta-glucoronidase, beta-lactamase, chloramphenicol acetyltransferase (“CAT”), and alkaline phosphatase (“AP”).
  • GFP green fluorescent protein
  • beta-galactosidase beta-galactosidase
  • beta-glucoronidase beta-lactamase
  • CAT chloramphenicol acetyltransferase
  • AP alkaline phosphatase Table 1 below lists various reporter genes and the properties of the products of the reporter genes that can be assayed
  • a reporter gene utilized in the reporter constructs is easily assayed and has an activity which is not normally found in the cell or organism of interest.
  • TABLE 1 Reporter Genes and the Properties of the Reporter Gene Products Reporter Gene Protein Activity & Measurement CAT (chloramphenicol acetyltransferase) Transfers radioactive acetyl groups to chloramphenicol or detection by thin layer chromatography and autoradiography
  • GAL beta-galactosidase
  • GUS beta-glucuronidase Hydrolyzes colorless glucuronides to yield colored products.
  • LUC luciferase Oxidizes luciferin, emitting photons GFP (green fluorescent protein) Fluorescent protein without substrate SEAP (secreted alkaline phosphatase) Luminescence reaction with suitable substrates or with substrates that generate chromophores HRP (horseradish peroxidase) In the presence of hydrogen oxide, oxidation of 3,3',5,5'-tetramethylbenzidine to form a colored complex AP (alkaline phosphatase) Luminescence reaction with suitable substrates or with substrates that generate chromophores
  • Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been used for real-time, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms (reviewed by Greer & Szalay, 2002, Luminescence 17(1):43-74 ).
  • luciferase is intended to embrace all luciferases, or recombinant enzymes derived from luciferases which have luciferase activity.
  • the luciferase genes from fireflies have been well characterized, for example, from the Photinus and Luciola species (see, e.g ., International Patent Publication No. WO 95/25798 for Photinus pyralis, European Patent Application No. EP 0 524 448 for Luciola cruciata and Luciola lateralis, and Devine et al., 1993, Biochim. Biophys. Acta 1173(2):121-132 for Luciola mingrelica ).
  • eucaryotic luciferase genes include, but are not limited to, the click beetle ( Photinus plagiophthalamus, see, e.g., Wood et al., 1989, Science 244:700-702 ), the sea panzy ( Renilla reniformis, see, e.g ., Lorenz et al., 1991, Proc Natl Acad Sci U S A 88(10):4438-4442 ), and the glow worm (Lampyris noctiluca, see e.g ., Sula-Newby et al., 1996, Biochem J. 313:761-767 ).
  • the click beetle Photinus plagiophthalamus, see, e.g., Wood et al., 1989, Science 244:700-702
  • the sea panzy Renilla reniformis, see, e.g ., Lorenz et al., 1991, Proc Natl Acad Sci
  • the click beetle is unusual in that different members of the species emit bioluminescence of different colors, which emit light at 546 nm (green), 560 nm (yellow-green), 578 nm (yellow) and 593 nm (orange) (see, e.g , U.S. Patent Nos. 6,475,719 ; 6,342,379 ; and 6,217,847 , the disclosures of which are incorporated by reference in their entireties).
  • Bacterial luciferin-luciferase systems include, but are not limited to, the bacterial lux genes of terrestrial Photorhabdus luminescens (see, e.g ., Manukhov et al., 2000, Genetika 36(3):322-30 ) and marine bacteria Vibrio fischeri and Vibrio harveyi (see, e.g ., Miyamoto et al., 1988, J Biol Chem. 263(26):13393-9 , and Cohn et al., 1983, Proc Natl Acad Sci USA., 80(1):120-3 , respectively).
  • the luciferases encompassed by the present invention also includes the mutant luciferases described in U.S. Patent No. 6,265,177 to Squirrell et al. , which is hereby incorporated by reference in its entirety.
  • the luciferase is a firefly luciferase, a renilla luciferase, or a click beetle luciferase, as described in any one of the references listed supra, the disclosures of which are incorporated by reference in their entireties.
  • Green fluorescent protein is a 238 amino acid protein with amino acid residues 65 to 67 involved in the formation of the chromophore which does not require additional substrates or cofactors to fluoresce (see, e.g ., Prasher et al., 1992, Gene 111:229-233 ; Yang et al., 1996, Nature Biotechnol. 14:1252-1256 ; and Cody et al., 1993, Biochemistry 32:1212-1218 ).
  • GFP green fluorescent protein
  • GFP includes green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, and blue fluorescent protein.
  • the native gene for GFP was cloned from the bioluminescent jellyfish Aequorea victoria (see, e.g ., Morin et al., 1972, J. Cell Physiol. 77:313-318 ). Wild type GFP has a major excitation peak at 395 nm and a minor excitation peak at 470 nm.
  • the absorption peak at 470 nm allows the monitoring of GFP levels using standard fluorescein isothiocyanate (FITC) filter sets.
  • Mutants of the GFP gene have been found useful to enhance expression and to modify excitation and fluorescence.
  • mutant GFPs with alanine, glycine, isoleucine, or threonine substituted for serine at position 65 result in mutant GFPs with shifts in excitation maxima and greater fluorescence than wild type protein when excited at 488 nm (see, e . g ., Heim et al., 1995, Nature 373:663-664 ; U.S. Patent No.
  • GFPs are isolated from organisms other than the jellyfish, such as, but not limited to, the sea pansy, Renilla reriformis.
  • GFPs in E. coli and C. elegans are described in U.S. Patent No. 6,251,384 to Tan et al. , which is incorporated by reference in its entirety.
  • the expression of GFP in plant cells is discussed in Hu & Cheng, 1995, FEBS Lett 369:331-33 , and GFP expression in Drosophila is described in Davis et al., 1995, Dev. Biology 170:726-729 .
  • Beta galactosidase is an enzyme that catalyzes the hydrolysis ofbeta-galactosides, including lactose, and the galactoside analogs o-nitrophenyl-beta-D-galactopyranoside ("ONPG”) and chlorophenol red-beta-D-galactopyranoside (“CPRG”) (see, e.g ., Nielsen et al., 1983 Proc Natl Acad Sci USA 80(17):5198-5202 ; Eustice et al., 1991, Biotechniques 11:739-742 ; and Henderson et al., 1986, Clin. Chem. 32:1637-1641 ).
  • ONPG o-nitrophenyl-beta-D-galactopyranoside
  • CPRG chlorophenol red-beta-D-galactopyranoside
  • beta-gal gene functions well as a reporter gene because the protein product is extremely stable, resistant to proteolytic degradation in cellular lysates, and easily assayed.
  • ONPG is used as the substrate, beta-gal activity can be quantitated with a spectrophotometer or microplate reader.
  • beta galactosidase or "beta-gal” is intended to embrace all beta-gals, including lacZ gene products, or recombinant enzymes derived from beta-gals which have beta-gal activity.
  • the beta-gal gene functions well as a reporter gene because the protein product is extremely stable, resistant to proteolytic degradation in cellular lysates, and easily assayed.
  • ONPG is the substrate
  • beta-gal activity can be quantitated with a spectrophotometer or microplate reader to determine the amount of ONPG converted at 420 nm.
  • beta-gal activity can be quantitated with a spectrophotometer or microplate reader to determine the amount of CPRG converted at 570 to 595 nm.
  • the beta-gal activity can be visually ascertained by plating bacterial cells transformed with a beta-gal construct onto plates containing Xga1 and IPTG. Bacterial colonies that are dark blue indicate the presence of high beta-gal activity and colonies that are varying shades of blue indicate varying levels of beta-gal activity.
  • Beta-glucuronidase catalyzes the hydrolysis of a very wide variety of beta-glucuronides, and, with much lower efficiency, hydrolyzes some beta-galacturonides. GUS is very stable, will tolerate many detergents and widely varying ionic conditions, has no cofactors, nor any ionic requirements, can be assayed at any physiological pH, with an optimum between 5.0 and 7.8, and is reasonably resistant to thermal inactivation (see, e.g ., U.S. Patent No. 5,268,463 , which is incorporated by reference in its entirety).
  • the GUS is derived from the Esherichia coli beta-glucuronidase gene.
  • the beta-glucuronidase encoding nucleic acid is homologous to the E. coli beta-glucuronidase gene and/or may be derived from another organism or species.
  • GUS activity can be assayed either by fluorescence or spectrometry, or any other method described in U.S. Patent No. 5,268,463 , the disclosure of which is incorporated by reference in its entirety.
  • 4-trifluoromethylumbelliferyl beta-D-glucuronide is a very sensitive substrate for GUS. The fluorescence maximum is close to 500 mm--bluish green, where very few plant compounds fluoresce or absorb. 4-trifluoromethylumbelliferyl beta-D-glucuronide also fluoresces much more strongly near neutral pH, allowing continuous assays to be performed more readily than with MUG.
  • 4-trifluoromethylumbelliferyl beta-D-glucuronide can be used as a fluorescent indicator in vivo .
  • the spectrophotometric assay is very straightforward and moderately sensitive ( Jefferson et al., 1986, Proc. Natl. Acad. Sci. USA 86:8447-8451 ).
  • a preferred substrate for spectrophotometric measurement is p-nitrophenyl beta-D-glucuronide, which when cleaved by GUS releases the chromophore p-nitrophenol. At a pH greater than its pK a (around 7.15) the ionized chromophore absorbs light at 400-420 nm, giving a yellow color.
  • Chloramphenicol acetyl transferase is commonly used as a reporter gene in mammalian cell systems because mammalian cells do not have detectable levels of CAT activity.
  • the assay for CAT involves incubating cellular extracts with radiolabeled chloramphenicol and appropriate co-factors, separating the starting materials from the product by, for example, thin layer chromatography ("TLC”), followed by scintillation counting (see, e.g ., U.S. Patent No. 5,726,041 , which is hereby incorporated by reference in its entirety).
  • chloramphenicol acetyltransferase or "CAT” is intended to embrace all CATs, or recombinant enzymes derived from CAT which have CAT activity. While it is preferable that a reporter system which does not require cell processing, radioisotopes, and chromatographic separations would be more amenable to high through-put screening, CAT as a reporter gene may be preferable in situations when stability of the reporter gene is important. For example, the CAT reporter protein has an in vivo half life of about 50 hours, which is advantageous when an accumulative versus a dynamic change type of result is desired.
  • the secreted alkaline phosphatase (“SEAP”) enzyme is a truncated form of alkaline phosphatase, in which the cleavage of the transmembrane domain of the protein allows it to be secreted from the cells into the surrounding media.
  • SEAP secreted alkaline phosphatase
  • the alkaline phosphatase is isolated from human placenta.
  • SEAP secreted alkaline phosphatase
  • SEAP activity can be detected by a variety of methods including, but not limited to, measurement of catalysis of a fluorescent substrate, immunoprecipitation, HPLC, and radiometric detection. The luminescent method is preferred due to its increased sensitivity over calorimetric detection methods.
  • the advantages of using SEAP is that a cell lysis step is not required since the SEAP protein is secreted out of the cell, which facilitates the automation of sampling and assay procedures.
  • nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease may be inserted into the coding region of a reporter gene such that the mRNA coding the reporter gene out of frame utilizing well-known molecular biology techniques.
  • a nucleotide sequence comprising a bulge-helix-bulge structure or a mature domain of a precursor tRNA may be inserted into the coding region of a reporter gene such that the mRNA coding the reporter gene out of frame.
  • nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease may be inserted into the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated regions of a reporter gene construct.
  • a nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease may comprise 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, or more.
  • the nucleotide sequence is at least 10 nucleotides in length.
  • a tRNA intron is inserted within the open reading frame of a reporter gene.
  • two, three, four, five or more tRNA introns are inserted within the open reading frame of a reporter gene.
  • a tRNA intron is inserted within the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated region of a reporter gene construct.
  • two, three, four, five or more tRNA introns are inserted within the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated region of a reporter gene construct.
  • the tRNA intron may comprise a bulge-helix-bulge conformation.
  • a reporter gene containing a tRNA intron may be produced by any method well-known to one of skill in the art.
  • the reporter gene containing a tRNA intron may be chemically synthesized using phosphoramidite or other solution or solid-phase methods.
  • phosphoramidite or other solution or solid-phase methods.
  • Detailed descriptions of the chemistry used to form polynucleotides by the phosphoramidite method are well known ( see, e.g., Caruthers et al., U.S. Pat. Nos.
  • such purification techniques include, but are not limited to, reverse-phase high-performance liquid chromatography ("reverse-phase HPLC”), fast performance liquid chromatography (“FPLC”), and gel purification.
  • Reverse-phase HPLC reverse-phase high-performance liquid chromatography
  • FPLC fast performance liquid chromatography
  • gel purification Methods for labeling the substrate with a fluorescent acceptor moiety, a fluorescent donor moiety and/or quencher are well-known in the art (see, e.g. , U.S. Patent Nos.
  • the nucleotide sequence coding for a reporter gene and the nucleotide sequence coding for a tRNA intron can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • the necessary transcriptional and translational signals can also be supplied by the reporter gene.
  • host-vector systems may be utilized to express the reporter gene. These include, but are not limited to, mammalian cell systems infected with virus (e.g ., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g.
  • baculovirus containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA; and stable cell lines generated by transformation using a selectable marker.
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA; and stable cell lines generated by transformation using a selectable marker.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric nucleic acid consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of the reporter gene construct may be regulated by a second nucleic acid sequence so that the reporter gene is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a reporter gene construct may be controlled by any promoter/enhancer element known in the art, such as a constitutive promoter, a tissue-specific promoter, or an inducible promoter.
  • promoters which may be used to control gene expression include, but are not limited to, the SV40 early promoter region ( Bemoist & Chambon, 1981, Nature 290:304-310 ), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus ( Yamamoto et al., 1980, Cell 22:787-797 ), the herpes thymidine kinase promoter ( Wagner et al., 1981, Proc. Natl. Acad. Sci U.S.A.
  • the regulatory sequences of the metallothionein gene ( Brinster et al., 1982, Nature 296:39-42 ); prokaryotic expression vectors such as the ⁇ -lactamase promoter ( Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731 ), or the tac promoter ( DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
  • promoter of the photosynthetic enzyme ribulose biphosphate carboxylase ( Herrera-Estrella et al., 1984, Nature 310:115-120 ); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells ( Swift et al., 1984, Cell 38:639-646 ; Ornitz et al., 1986, Cold Spring Harbor Symp.
  • mice mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells
  • Leder et al., 1986, Cell 45:485-495 albumin gene control region which is active in liver
  • albumin gene control region which is active in liver
  • alpha-fetoprotein gene control region which is active in liver
  • Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648 Hammer et al., 1987, Science 235:53-58
  • alpha 1-antitrypsin gene control region which is active in the liver
  • beta-globin gene control region which is active in myeloid cells ( Mogram et al., 1985, Nature 315:338-340 ; Kollias et al., 1986, Cell 46:89-94 ; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain ( Readhead et al., 1987, Cell 48:703-712 ); myosin light chain-2 gene control region which is active in skeletal muscle ( Sani, 1985, Nature 314:283-286 ), and gonadotropic releasing hormone gene control region which is active in the hypothalamus ( Mason et al., 1986, Science 234:1372-1378 ).
  • a vector in a specific embodiment, comprises a promoter operably linked to a reporter gene, one or more origins of replication, and, optionally, one or more selectable markers (e.g. , an antibiotic resistance gene).
  • the vectors are CMV vectors, T7 vectors, lac vectors, pCEP4 vectors, 5.0/F vectors, or vectors with a tetracycline-regulated promoter (e . g ., pcDNA TM 5/FRT/TO from Invitrogen
  • Expression vectors containing the reporter gene construct of the present invention can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" nucleic acid functions, (c) expression of inserted sequences, and (d) sequencing.
  • first approach the presence of the reporter gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the inserted reporter gene.
  • second approach the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" nucleic acid functions (e.g.
  • recombinant expression vectors can be identified by assaying the reporter gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the particular reporter gene.
  • the reporter gene constructs are cloned into stable cell line expression vectors.
  • the stable cell line expression vector contains a site specific genomic integration site.
  • the reporter gene construct is cloned into an episomal mammalian expression vector.
  • a host cell is transformed or transfected with the vector of interest.
  • the use of stable transformants is preferred.
  • the host cell is a mammalian cell.
  • the host cell is a human cell.
  • the host cells are primary cells isolated from a tissue or other biological sample of interest. Host cells that can be used in the methods of the present invention include, but are not limited to, hybridomas, pre-B cells, 293 cells, 293T cells, HeLa cells, HepG2 cells, K562 cells, 3T3 cells.
  • the host cells are immortalized cell lines derived from a source, e.g. , a tissue. Other host cells that can be used in the present invention include, but are not limited to, virally-infected cells.
  • Transformation may be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide.
  • the transformation procedure used depends upon the host to be transformed.
  • Mammalian transformations (i.e. , transfections) by direct uptake may be conducted using the calcium phosphate precipitation method of Graham & Van der Eb, 1978, Virol. 52:546 , or the various known modifications thereof.
  • stable cell lines containing the constructs of interest are generated for high throughput screening.
  • Such stable cells lines may be generated by introducing a reporter gene construct comprising a selectable marker, allowing the cells to grow for 1-2 days in an enriched medium, and then growing the cells on a selective medium.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase ( Wigler, et al., 1977, Cell 11:223 ), hypoxanthine-guanine phosphoribosyltransferase ( Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026 ), and adenine phosphoribosyltransferase ( Lowy, et al., 1980, Cell 22:817 ) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • anti-metabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate ( Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567 ; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527 ); gpt, which confers resistance to mycophenolic acid ( Mulligan & Berg, 1981, Proc. Natal. Acad. Sci. USA 78:2072 ); neo, which confers resistance to the aminoglycoside G-418 ( Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1 ); and hygro, which confers resistance to hygromycin ( Santerre, et al., 1984, Gene 30:147 ) genes.
  • the invention provides for the translation of the reporter gene constructs in a cell-free system.
  • Techniques for practicing this specific aspect of this invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and recombinant DNA manipulation and production, which are routinely practiced by one of skill in the art. See, e.g., Sambrook, 1989, Molecular Cloning, A Laboratory Manual, Second Editi on; DNA Cloning, Volumes I and II (Glover, Ed. 1985 ); and Transcription and Translation (Hames & Higgins, Eds. 1984 ).
  • a cell extract utilized in accordance with the invention may be an S1 extract (i.e., the supernatant from a 1,000 x g spin) to an S500 extract ( i.e. , the supernatant from a 500,000 x g spin), preferably an S10 extract (i.e., the supernatant from a 10,000 x g spin) to an S250 extract ( i.e., the supernatant from a 250,000 x g spin).
  • a cell extract utilized in accordance with the invention is an S50 extract (i.e. , the supernatant from a 50,000 x g spin) to an S100 extract (i.e., the supernatant from a 100,000 x g spin).
  • the cell-free translation extract may be isolated from cells of any species origin.
  • the cell-free translation extract may be isolated from human cells, cultured mouse cells, cultured rat cells, Chinese hamster ovary (CHO) cells, Xenopus oocytes, rabbit reticulocytes, wheat germ, or rye embryo (see, e.g. , Krieg & Melton, 1984, Nature 308:203 and Dignam et al., 1990 Methods Enzymol. 182:194-203 ).
  • the cell-free translation extract e.g. , rabbit reticulocyte lysates and wheat germ extract, can be purchased from, e.g. , Promega, (Madison, WI).
  • the cell-free extract is an extract isolated from human cells.
  • the human cells are HeLa cells.
  • Animalia tRNA splicing endonuclease or a subunit thereof, preferably mammalian, more preferably human, can be expressed and purified by any method known to the skilled artisan.
  • Animalia tRNA splicing endonuclease can be expressed by recombinant DNA technology.
  • the animalia tRNA splicing endonuclease is fused to a peptide tag to facilitate purification of the animalia tRNA splicing endonuclease.
  • the endogenous animalia tRNA splicing endonuclease is purified.
  • recombinant human tRNA splicing endonuclease is purified and used with the methods of the invention.
  • partially purified human tRNA splicing endonuclease from any human cell source is used with the methods of the invention.
  • an animalia tRNA splicing endonuclease subunit is encoded by a specific nucleotide sequence which is to be transcribed and translated. The nucleotide sequence is inserted into an expression vector for propagation and expression in recombinant cells.
  • An animalia tRNA splicing endonuclease is a heterotetramer, each of the four subunits may be expressed together in the same cell or separately in different cells; the subunits isolated and then combined to produce tRNA splicing endonuclease.
  • the animalia tRNA splicing endonuclease subunits are expressed in the same cell and the functional tRNA splicing endonuclease is isolated from the cell.
  • An expression construct refers to a nucleotide sequence encoding one, two, three or four animalia tRNA splicing endonuclease subunits (preferably, human tRNA splicing endonuclease subunits) operably linked to one or more regulatory regions or enhancer/promoter sequences which enables the expression of animalia tRNA splicing endonuclease subunits in an appropriate host cell.
  • animalia tRNA splicing endonuclease subunits preferably, human tRNA splicing endonuclease subunits
  • “Operably linked” refers to an association in which the regulatory regions and the nucleotide sequence encoding an animalia tRNA splicing endonuclease subunit that is to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.
  • the regulatory regions necessary for transcription of an animalia tRNA splicing endonuclease subunit can be provided by the expression vector.
  • cellular transcriptional factors such as RNA polymerase
  • RNA polymerase will bind to the regulatory regions on the expression construct to effect transcription of an animalia tRNA splicing endonuclease subunit in the host organism.
  • the precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence.
  • Such regulatory regions may include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
  • Constitutive, tissue-specific and/or inducible regulatory regions may be used for expression of an animalia tRNA splicing endonuclease subunit. It may be desirable to use inducible promoters when the conditions optimal for growth of the host cells and the conditions for high level expression of the animalia tRNA splicing endonuclease subunit are different. Examples of useful regulatory regions are provided below.
  • linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art ( Wu et al., 1987, Methods in Enzymol 152:343-349 ). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.
  • An expression construct comprising a sequence encoding an animalia tRNA splicing endonuclease subunit operably linked to regulatory regions (enhancer/promoter sequences) can be directly introduced into appropriate host cells for expression and production of an animalia tRNA splicing endonuclease subunit without further cloning.
  • the expression constructs can also contain DNA sequences that facilitate integration of the sequence encoding an animalia tRNA splicing endonuclease subunit into the genome of the host cell, e.g., via homologous recombination. In this instance, it is not necessary to employ an expression vector comprising a replication origin suitable for appropriate host cells in order to propagate and express an animalia tRNA splicing endonuclease subunit in the host cells.
  • expression vectors may be used in the present invention which include, but are not limited to, plasmids, cosmids, phage, phagemids, or modified viruses.
  • expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the sequence encoding the animalia tRNA splicing endonuclease subunit, and one or more selection markers.
  • the expression vector must be used with a compatible host cell which may be derived from a prokaryotic or an eukaryotic organism including but not limited to bacteria, yeasts, insects, mammals, and humans.
  • Non-limiting examples of regulatory regions that can be used for expression in E . coli may include but not limited to lac, trp, lpp, phoA, recA, tac, T3, T7 and ⁇ P L ( Makrides, 1996, Microbiol Rev, 60:512-538 ).
  • Non-limiting examples of prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gt11 ( Huynh et al., 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach (D. Glover, ed.), pp.
  • a potential drawback of a prokaryotic host-vector system is the inability to perform many of the post-translational processing of mammalian cells.
  • an eukaryotic host-vector system is preferred, a mammalian host-vector system is more preferred, and a human host-vector system is the most preferred.
  • a variety of regulatory regions can be used, for example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.
  • CMV cytomegalovirus
  • RSV-LTR Rous sarcoma virus long terminal repeat
  • Inducible promoters that may be useful in mammalian cells include but are not limited to those associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), ⁇ -interferon gene, and hsp70 gene ( Williams et al., 1989, Cancer Res.
  • the expression vector may contain selectable or screenable marker genes for initially isolating, identifying or tracking host cells that contain DNA encoding the elected animalia tRNA splicing endonuclease subunit.
  • selectable or screenable marker genes for initially isolating, identifying or tracking host cells that contain DNA encoding the elected animalia tRNA splicing endonuclease subunit.
  • stable expression in mammalian cells is preferred.
  • a number of selection systems may be used for mammalian cells, including but not limited to the Herpes simplex virus thymidine kinase ( Wigler et al., 1977, Cell 11:223 ), hypoxanthine-guanine phosphoribosyltransferase ( Szybalski and Szybalski, 1962, Proc. Natl.
  • adenine phosphoribosyltransferase genes can be employed in tk - , hgprt - or aprt - cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate ( Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567 ; O'Hare et al., 1981, Proc. Natl. Acad. Sci.
  • Generating a fusion protein comprising a peptide tag and an animalia tRNA splicing endonuclease subunit can aid the purification of the animalia tRNA splicing endonuclease subunit.
  • the animalia tRNA splicing endonuclease subunit is a mammalian tRNA splicing endonuclease subunit.
  • the animalia tRNA splicing endonuclease is a human animalia tRNA splicing endonuclease subunit.
  • a fusion protein comprising a peptide and an animalia tRNA splicing endonuclease subunit can be made by ligating the nucleotide sequence encoding the animalia tRNA splicing endonuclease subunit to the sequence encoding the peptide tag in the proper reading frame. Care should be taken to ensure that the modified gene remains within the same translational reading frame, uninterrupted by translational stop signals and/or spurious messenger RNA splicing signals.
  • the peptide tag may be fused to the amino terminal or to the carboxyl terminal of an animalia tRNA splicing endonuclease subunit.
  • the precise site at which the fusion is made is not critical. The optimal site can be determined by routine experimentation.
  • a variety of peptide tags known in the art may be conjugated to an animalia tRNA splicing endonuclease subunit including, but not limited to the immunoglobulin constant regions, polyhistidine sequence ( Petty, 1996, Metal-chelate affinity chromatography, in Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience ), glutathione S-transferase (GST; Smith, 1993, Methods Mol. Cell Bio. 4:220-229 ), the E . coli maltose binding protein ( Guan et al., 1987, Gene 67:21-30 ), various cellulose binding domains ( U.S.
  • peptide tags As will be appreciated by those skilled in the art, many methods can be used to obtain the coding region of the above-mentioned peptide tags, including but not limited to, DNA cloning, DNA amplification, and synthetic methods. Some of the peptide tags and reagents for their detection and isolation are available commercially.
  • the polyhistidine tag conjugated to an animalia tRNA splicing endonuclease subunit has at least 6, at least 8, at least 10 or at least 10 histidines. In a preferred embodiment, the polyhistidine tag conjugated to an animalia tRNA splicing endonuclease subunit has 8 histidines.
  • an animalia tRNA splicing endonuclease subunit can be labeled with more than one peptide.
  • an animalia tRNA splicing endonuclease subunit is labeled with a peptide tag consisting of 8 histidines and a Flag epitope tag.
  • different subunits of an animalia tRNA splicing endonuclease can be conjugated to different peptide tags.
  • Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, (see, for example, Kriegler M. in “Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990 ), such as monkey kidney cell line transformed by SV40 (COS-7, ATCC Accession No. CRL 1651); human embryonic kidney cell lines (293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 36:59, 1977 ; baby hamster kidney cells (BHK, ATCC Accession No. CCL 10); chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin. Proc. Natl.
  • mouse sertoli cells Mather, Biol. Reprod. 23:243-251, 1980 ); mouse fibroblast cells (NIH-3T3), monkey kidney cells (CVI ATCC Accession No. CCL 70); african green monkey kidney cells (VERO-76, ATCC Accession No. CRL-1587); human cervical carcinoma cells (HELA, ATCC Accession No. CCL 2); canine kidney cells (MDCK, ATCC Accession No. CCL 34); buffalo rat liver cells (BRL 3A, ATCC Accession No. CRL 1442); human lung cells (W138, ATCC Accession No. CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC Accession No. CCL51).
  • a number of viral-based expression systems may also be utilized with mammalian cells to produce an animalia tRNA splicing endonuclease subunit.
  • Vectors using DNA virus backbones have been derived from simian virus 40 (SV40) ( Harner et al., 1979, Cell 17:725 ), adenovirus ( Van Doren et al., 1984, Mol Cell Biol 4:1653 ), adeno-associated virus ( McLaughlin et al., 1988, J Virol 62:1963 ), and bovine papillomas virus ( Zinn et al., 1982, Proc Natl Acad Sci 79:4897 ).
  • SV40 simian virus 40
  • adenovirus Van Doren et al., 1984, Mol Cell Biol 4:1653
  • McLaughlin et al., 1988, J Virol 62:1963 adeno-associated virus
  • bovine papillomas virus Zinn e
  • the donor DNA sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts.
  • a non-essential region of the viral genome e.g., region E1 or E3
  • yeast a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts).
  • Saccharomyces cerevisiae bakeer's yeast
  • Schizosaccharomyces pombe Schizosaccharomyces pombe
  • Pichia pastoris Pichia pastoris
  • Hansenula polymorpha methylotropic yeasts
  • Autographa california nuclear polyhidrosis virus (AcNPV) a baculovirus
  • AcNPV Autographa california nuclear polyhidrosis virus
  • the sequences encoding an animalia tRNA splicing endonuclease subunit may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • AcNPV promoter for example the polyhedrin promoter
  • any of the cloning and expression vectors described herein may be synthesized and assembled from known DNA sequences by well known techniques in the art.
  • the regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic.
  • Some vectors and host cells may be obtained commercially. Non-limiting examples of useful vectors are described in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience , which is incorporated herein by reference; and the catalogs of commercial suppliers such as Clontech Laboratories, Stratagene Inc., and Invitrogen, Inc.
  • Expression constructs containing a cloned nucleotide sequence encoding an animalia tRNA splicing endonuclease subunit can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, bacterial transformation ( Hanahan, 1985, in DNA Cloning, A Practical Approach, 1:109-136 ), and for eukaryotic cells, calcium phosphate mediated transfection ( Wigler et al., 1977, Cell 11:223-232 ), liposome-mediated transfection ( Schaefer-Ridder et al., 1982, Science 215:166-168 ), electroporation ( Wolff et al., 1987, Proc Natl Acad Sci 84:3344 ), and microinjection ( Cappechi, 1980, Cell 22:479-488 ).
  • Cell lines that stably express an animalia tRNA splicing endonuclease subunit may be engineered by using a vector that contains a selectable marker.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while an animalia tRNA splicing endonuclease subunit is expressed continuously.
  • an animalia human tRNA splicing endonuclease subunit is transfected stably in 293T cells (ATCC Accession No. CRL-11268).
  • an animalia tRNA splicing endonuclease subunit or the animalia tRNA splicing endonulcease can be recovered and purified from recombinant cell cultures by known methods, including ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, immunoaffinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • the animalia tRNA splicing endonuclease subunit or animalia tRNA splicing endonuclease is a mammalian tRNA splicing endonuclease subunit or a mammalian tRNA splicing endonuclease, respectively.
  • the animalia tRNA splicing endonuclease subunit or animalia tRNA splicing endonuclease is a human tRNA splicing endonuclease subunit or a human tRNA splicing endonuclease, respectively.
  • total protein has to be prepared from the cell culture. This procedure comprises collection, washing and lysis of said cells and is well known to the skilled artisan.
  • a recombinant animalia tRNA splicing endonuclease subunit fused to a peptide tag may be purified based on the properties of the peptide tag.
  • One approach is based on specific molecular interactions between a tag and its binding partner.
  • the other approach relies on the immunospecific binding of an antibody to an epitope present on the tag or on the protein which is to be purified.
  • affinity chromatography well known in the art is generally applicable to both of these approaches.
  • fractions can be collected and tested for the presence of the animalia tRNA splicing endonuclease and/or for the presence of the peptide tag.
  • the fractions are tested for tRNA splicing endonuclease activity.
  • the fractions with tRNA splicing endonuclease activity levels over a certain threshold level can be pooled.
  • a method that is generally applicable to purifying an animalia tRNA splicing endonuclease subunit that are fused to the constant regions of immunoglobulin is protein A affinity chromatography, a technique that is well known in the art.
  • Staphylococcus protein A is a 42 kD polypeptide that binds specifically to a region located between the second and third constant regions of heavy chain immunoglobulins. Because of the Fc domains of different classes, subclasses and species of immunoglobulins, affinity of protein A for human Fc regions is strong, but may vary among species. Subclasses that are less preferred include human IgG-3, and most rat subclasses.
  • protein G (of Streptococci) may be used in place of protein A in the purification.
  • Protein-A sepharose (Pharmacia or Biorad) is a commonly used solid phase for affinity purification of antibodies, and can be used essentially in the same manner for the purification of an animalia tRNA splicing endonuclease subunit fused to an immunoglobulin Fc fragment.
  • Bound animalia tRNA splicing endonuclease subunit-Fc fusion protein can be eluted by various buffer systems known in the art, including a succession of citrate, acetate and glycine-HCl buffers which gradually lowers the pH.
  • the recombinant cells also produce antibodies which will be co-purified with the human tRNA splicing endonuclease subunit. See, for example, Langone, 1982, J. Immunol. meth. 51:3 ; Wilchek et al., 1982, Biochem. Intl. 4:629 ; Sjobring et al., 1991, J. Biol. Chem. 26:399; page 617-618 , in Antibodies A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor laboratory, 1988 .
  • a polyhistidine tag may be used, in which case, an animalia tRNA splicing endonuclease subunit can be purified by metal chelate chromatography.
  • the polyhistidine tag usually a sequence of six histidines, has a high affinity for divalent metal ions, such as nickel ions (Ni 2+ ), which can be immobilized on a solid phase, such as nitrilotriacetic acid-matrices.
  • Polyhistidine has a well characterized affinity for Ni 2+ -NTA-agarose, and can be eluted with either of two mild treatments: imidazole (0.1-0.2 M) will effectively compete with the resin for binding sites; or lowering the pH just below 6.0 will protonate the histidine sidechains and disrupt the binding.
  • the purification method comprises loading the cell culture lysate onto the Ni 2+ -NTA-agarose column, washing the contaminants through, and eluting the animalia tRNA splicing endonuclease subunit with imidazole or weak acid.
  • Ni 2+ -NTA-agarose can be obtained from commercial suppliers such as Sigma (St. Louis) and Qiagen.
  • Antibodies that recognize the polyhistidine tag are also available which can be used to detect and quantitate the human tRNA splicing endonuclease subunit.
  • GST glutathione-S-transferase
  • an animalia tRNA splicing endonuclease subunit-GST fusion protein expressed in a prokaryotic host cell, such as E. coli can be purified from the cell culture lysate by absorption with glutathione agarose beads, followed by elution in the presence of free reduced glutathione at neutral pH. Since GST is known to form dimers under certain conditions, dimeric animalia tRNA splicing endonuclease subunit may be obtained. See, Smith, 1993, Methods Mol. Cell Bio. 4:220-229 .
  • MBP maltose binding protein
  • An animalia tRNA splicing endonuclease subunit fused to MBP binds to amylose resin while contaminants are washed away.
  • the bound animalia tRNA splicing endonuclease subunit-MBP fusion is eluted from the amylose resin by maltose. See, for example, Guan et al., 1987, Gene 67:21-30 .
  • the second approach for purifying an animalia tRNA splicing endonuclease subunit or animalia tRNA splicing endonuclease is applicable to peptide tags that contain an epitope for which polyclonal or monoclonal antibodies are available. It is also applicable if polyclonal or monoclonal antibodies specific to an animalia tRNA splicing endonuclease subunit or the animalia tRNA splicing endonuclease are available.
  • the invention relates to the expression and purification of the human tRNA splicing endonuclease subunits Hs Sen2p and Hs Sen34p (see Table 2).
  • Oligonucleotides complementary to the 5' and 3' ends of the open reading frames of the animalia tRNA splicing endonuclease subunits can be used to PCR amplify the open reading frames encoding the animalia tRNA splicing endonuclease.
  • the invention also relates to the expression and purification of an Hs Sen 2p variant ("Hs Sen 2 var.”).
  • the Hs Sen 2 var. is a splice variant of Hs Sen2 lacking exon 8 of the genomic DNA sequence for Human Sen 2.
  • Figure 2 depicts an amino acid sequence alignment of the amino acid sequences of the two human Sen 2 subunits (i.e., Hs Sen2 and Hs Sen 2 var.) and the amino acid sequence of the yeast subunit Sc Sen 2p.
  • the sequence alignment reveals a high degree of similarity in the YRGGY motif (SEQ ID NO: 4), the active site for the 5' splice site of yeast (Sc Sen 2p) and archael (not shown) tRNA splicing endonuclease.
  • the Hs Sen 2 var. lacks the putative transmembrane domain found in the Hs Sen 2 endonuclease, which may affect the localization of the Hs Sen2 var. in an animalia cell.
  • the Hs Sen2 var. catalyzes the endonucleolytic cleavage of substrates other than those containing tRNA introns. In other embodiments, the Hs Sen2 var. catalyzes the endonucleolytic cleavage of substrates containing tRNA introns. In yet other embodiments, the Hs Sen2 var. catalyzes the endonucleolytic cleavage of substrates containing tRNA introns and substrates that do not contain tRNA introns.
  • the human subunits including, but not limited to, Hs Sen2, Hs Sen2 var and Hs Sen 34, can be utilized in accordance with the methods of the invention.
  • the Hs Sen 2 subunit is utilized in accordance with the methods of the invention.
  • the Hs Sen 2 var. subunit is utilized in accordance with the methods of the invention.
  • the Hs Sen 34 subunit is utilized in accordance with the methods of the invention.
  • Hs Sen 2, Hs Sen 2 var., Hs Sen 34 or any combination thereof is utilized in accordance with the methods of the invention.
  • Fungal tRNA splicing endonuclease subunits in particular, the yeast tRNA splicing endonuclease subunits
  • the fungal tRNA splicing endonuclease in particular, yeast tRNA splicing endonuclease
  • a fungal tRNA splicing endonuclease subunit or the fungal tRNA splicing endonuclease can be purified by the methods discussed above for an animalia tRNA splicing endonuclease subunit or the animalia tRNA splicing endonuclease.
  • yeast tRNA splicing endonuclease or a subunit thereof is purified according to the procedure described in Trotta et al., 1997, Cell 89:849-858 .
  • Libraries screened using the methods of the present invention can comprise a variety of types of compounds.
  • libraries that can be screened in accordance with the methods of the invention include, but are not limited to, peptoids; random biooligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and small molecule libraries (preferably, small organic molecule libraries).
  • the compounds in the libraries screened are nucleic acid or peptide molecules.
  • peptide molecules can exist in a phage display library.
  • the types of compounds include, but are not limited to, peptide analogs including peptides comprising non-naturally occurring amino acids, e.g.
  • D-amino acids D-amino acids, phosphorous analogs of amino acids, such as ⁇ -amino phosphoric acids and ⁇ -amino phosphoric acids, or amino acids having non-peptide linkages, nucleic acid analogs such as phosphorothioates and PNAs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. Libraries of polypeptides or proteins can also be used in the assays of the invention.
  • the combinatorial libraries are small organic molecule libraries including, but not limited to, benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, and benzodiazepines.
  • the combinatorial libraries comprise peptoids; random bio-oligomers; benzodiazepines; diversomers such as hydantoins, benzodiazepines and dipeptides;, vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; or carbohydrate libraries.
  • Combinatorial libraries are themselves commercially available (see, e.g. , ComGenex, Princeton, New Jersey; Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Missouri; ChemStar, Ltd, Moscow, Russia; 3D Pharmaceuticals, Exton, Pennsylvania; Martek Biosciences, Columbia, Maryland; etc .).
  • the library is preselected so that the compounds of the library are more amenable for cellular uptake.
  • compounds are selected based on specific parameters such as, but not limited to, size, lipophilicity, hydrophilicity, and hydrogen bonding, which enhance the likelihood of compounds getting into the cells.
  • the compounds are analyzed by three-dimensional or four-dimensional computer computation programs.
  • the combinatorial compound library for use in accordance with the methods of the present invention may be synthesized.
  • synthetic methods directed toward the creation of large collections of small organic compounds, or libraries, which could be screened for pharmacological, biological or other activity.
  • the synthetic methods applied to create vast combinatorial libraries are performed in solution or in the solid phase, i.e., on a solid support.
  • Solid-phase synthesis makes it easier to conduct multi-step reactions and to drive reactions to completion with high yields because excess reagents can be easily added and washed away after each reaction step.
  • Solid-phase combinatorial synthesis also tends to improve isolation, purification and screening. However, the more traditional solution phase chemistry supports a wider variety of organic reactions than solid-phase chemistry.
  • Combinatorial compound libraries of the present invention may be synthesized using the apparatus described in U.S. Patent No. 6,190,619 to Kilcoin et.al., which is hereby incorporated by reference in its entirety.
  • U.S. Patent No. 6,190,619 discloses a synthesis apparatus capable of holding a plurality of reaction vessels for parallel synthesis of multiple discrete compounds or for combinatorial libraries of compounds.
  • the combinatorial compound library can be synthesized in solution.
  • the method disclosed in U.S. Patent No. 6,194,612 to Boger et al. which is hereby incorporated by reference in its entirety, features compounds useful as templates for solution phase synthesis of combinatorial libraries.
  • the template is designed to permit reaction products to be easily purified from unreacted reactants using liquid/liquid or solid/liquid extractions.
  • the compounds produced by combinatorial synthesis using the template will preferably be small organic molecules. Some compounds in the library may mimic the effects of non-peptides or peptides.
  • liquid phase synthesis does not require the use of specialized protocols for monitoring the individual steps of a multistep solid phase synthesis ( Egner et al., 1995, J.Org. Chem. 60:2652 ; Anderson et al., 1995, J. Org. Chem. 60:2650 ; Fitch et al., 1994, J. Org. Chem. 59:7955 ; Look et al., 1994, J. Org. Chem. 49:7588 ; Metzger et al., 1993, Angew. Chem., Int. Ed. Engl. 32:894 ; Youngquist et al., 1994, Rapid Commun.
  • Combinatorial compound libraries useful for the methods of the present invention can be synthesized on solid supports.
  • a split synthesis method a protocol of separating and mixing solid supports during the synthesis, is used to synthesize a library of compounds on solid supports (see e.g. , Lam et al., 1997, Chem. Rev. 97:41-448 ; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926 and references cited therein).
  • Each solid support in the final library has substantially one type of compound attached to its surface.
  • solid support is not limited to a specific type of solid support. Rather a large number of supports are available and are known to one skilled in the art. Solid supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, polystyrene beads, alumina gels, and polysaccharides. A suitable solid support may be selected on the basis of desired end use and suitability for various synthetic protocols. For example, for peptide synthesis, a solid support can be a resin such as p-methylbenzhydrylamine (pMBHA) resin (Peptides International, Louisville, KY), polystyrenes (e.g.
  • pMBHA p-methylbenzhydrylamine
  • PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.
  • chloromethylpolystyrene hydroxymethylpolystyrene and aminomethylpolystyrene
  • poly (dimethylacrylamide)-grafted styrene co-divinyl-benzene e.g. , POLYHIPE resin, obtained from Aminotech, Canada
  • polyamide resin obtained from Aminotech, Canada
  • polyamide resin obtained from Peninsula Laboratories
  • polystyrene resin grafted with polyethylene glycol e.g. , TENTAGEL or ARGOGEL, Bayer, Tubingen, Germany
  • polydimethylacrylamide resin obtained from Milligen/Biosearch, California
  • Sepharose Pharmacia, Sweden
  • compounds can be attached to solid supports via linkers.
  • Linkers can be integral and part of the solid support, or they may be nonintegral that are either synthesized on the solid support or attached thereto after synthesis.
  • Linkers are useful not only for providing points of compound attachment to the solid support, but also for allowing different groups of molecules to be cleaved from the solid support under different conditions, depending on the nature of the linker.
  • linkers can be, inter alia, electrophilically cleaved, nucleophilically cleaved, photocleavable, enzymatically cleaved, cleaved by metals, cleaved under reductive conditions or cleaved under oxidative conditions.
  • the compounds are cleaved from the solid support prior to high throughput screening of the compounds.
  • the compound is a small molecule.
  • in vitro assays can be used to identify and verify the ability of a compound to modulate the activity of an animalia tRNA splicing endonuclease. Multiple in vitro assays can be performed simultaneously or sequentially to assess the affect of a compound on the activity of an animalia tRNA splicing endonuclease. In a preferred embodiment, the in vitro assays described herein are performed in a high throughput format.
  • the animalia tRNA splicing endonuclease utilized in the assays described herein is a mammalian tRNA splicing endonuclease and more preferably, a human tRNA splicing endonuclease.
  • the cells are used to screen the library to identify compounds that modulate the activity of an animalia tRNA splicing endonuclease.
  • the reporter gene-based assays may be conducted by contacting a compound or a member of a library of compounds with a cell genetically engineered to contain a reporter gene construct comprising a reporter gene and a tRNA intron within the open reading frame of the reporter gene, or within the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated regions of the reporter gene construct, or within a mRNA splice site of the reporter gene; and measuring the expression of said reporter gene.
  • the alteration in reporter gene expression relative to a previously determined reference range, the absence of the compound or a control in such reporter-gene based assays indicates that a particular compound modulates the activity of an animalia tRNA splicing endonuclease.
  • An increase in reporter gene expression relative to a previously determined reference range, the absence of the compound or a control in such reporter-gene based assays indicates that a particular compound enhances the activity of an animalia tRNA splicing endonuclease.
  • a negative control e.g., PBS or another agent that is known to have no effect on the expression of the reporter gene
  • a positive control e.g., an agent that is known to have an effect on the expression of the reporter gene, preferably an agent that effects the activity of an animalia tRNA splicing endonuclease
  • the step of contacting a compound or a member of a library of compounds with an animalia cell genetically engineered to contain a reporter gene construct comprising a reporter gene and a tRNA intron within the open reading frame of the reporter gene, within the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated regions of the reporter gene construct or within a mRNA splice site may be conducted under physiologic conditions.
  • a compound or a member of a library of compounds is added to the cells in the presence of an aqueous solution.
  • the aqueous solution may comprise a buffer and a combination of salts, preferably approximating or mimicking physiologic conditions.
  • the aqueous solution may comprise a buffer, a combination of salts, and a detergent or a surfactant.
  • salts which may be used in the aqueous solution include, but not limited to, KCl, NaCl, and/or MgCl 2 .
  • the optimal concentration of each salt used in the aqueous solution is dependent on the cells and compounds used and can be determined using routine experimentation.
  • the step of contacting a compound or a member of a library of compounds with an animalia cell genetically engineered to contain the reporter gene construct may be performed for at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • the invention provides a method for identifying a compound that modulates animalia tRNA splicing endonuclease activity, said method comprising: (a) expressing a nucleic acid comprising a reporter gene in a cell, wherein the reporter gene comprises a tRNA intron; (b) contacting said cell with a member of a library of compounds; and (c) detecting the expression of said reporter gene, wherein a compound that modulates tRNA splicing endonuclease activity is identified if the expression of said reporter gene in the presence of a compound is altered relative to a previously determined reference range or the expression of said reporter gene in the absence of the compound or the presence of a control.
  • the invention provides a method for identifying a compound that modulates animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting a member of a library of compounds with a cell containing a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron; and (b) detecting the expression of said reporter gene, wherein a compound that modulates tRNA splicing endonuclease activity is identified if the expression of said reporter gene in the presence of a compound is altered relative to a previously determined reference range the expression of said reporter gene in the absence of said compound or the presence of a control.
  • reporter gene and/or activity of the protein encoded by the reporter gene in the cell-based reporter-gene assays may be detected by any technique well-known to one of skill in the art.
  • the expression of a reporter gene can be readily detected, e.g. , by quantifying the protein and/or RNA encoded by said gene.
  • Many methods standard in the art can be thus employed, including, but not limited to, immunoassays to detect and/or visualize gene expression (e.g.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1 % Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g. , EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody which recognizes the antigen to the cell lysate, incubating for a period of time (e.g.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1 % Trasylol
  • protein phosphatase and/or protease inhibitors e.g. , ED
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g. , 8%- 20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g. , PBS-Tween 20), blocking the membrane with primary antibody (the antibody which recognizes the antigen) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g.
  • a polyacrylamide gel e.g. , 8%- 20% SDS-PAGE depending on the molecular weight of the antigen
  • a membrane such as nitrocellulose, PVDF or nylon
  • blocking solution e.g., PBS with 3% BSA or non-fat milk
  • washing buffer
  • an antihuman antibody conjugated to an enzymatic substrate e.g. , horseradish peroxidase or alkaline phosphatase
  • radioactive molecule e.g. , 32 P or 125 I
  • blocking buffer washing the membrane in wash buffer, and detecting the presence of the antigen.
  • radioactive molecule e.g. , 32 P or 125 I
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding a primary antibody (which recognizes the antigen) conjugated to a detectable compound such as an enzymatic substrate (e.g. , horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a primary antibody which recognizes the antigen conjugated to a detectable compound
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the primary antibody) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well.
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1 .
  • luciferase beta-galactosidase
  • GUS beta-glucoronidase
  • CAT chloramphenicol acetyltransferase
  • AP alkaline phosphatase
  • the reaction products of luciferase, beta-galactosidase (“beta-gal”), and alkaline phosphatase (“AP”) are assayed by changes in light imaging (e.g. , luciferase), spectrophotometric absorbance (e.g. , beta-gal), or fluorescence (e.g. , AP).
  • Assays for changes in light output, absorbance, and/or fluorescence are easily adapted for high throughput screening.
  • beta-gal activity can be measured with a microplate reader.
  • Green fluorescent protein (“GFP”) activity can be measured by changes in fluorescence.
  • FACS fluorescence activated cell sorting
  • Alterations in the expression of a reporter gene may be determined by comparing the level of expression of the reporter gene to a negative control (e.g. , PBS or another agent that is known to have no effect on the expression of the reporter gene) and optionally, a positive control (e.g. , an agent that is known to have an effect on the expression of the reporter gene, preferably an agent that effects the activity of an animalia tRNA splicing endonuclease).
  • alterations in the expression of a reporter gene may be determined by comparing the level of expression of the reporter gene to a previously determined reference range.
  • a cell-free translation extract is generated or purchased, and a compound library is synthesized or purchased or both, the cell-free translation extract and nucleic acid are used to screen the library to identify compounds that modulate the activity of an animalia tRNA splicing endonuclease.
  • the reporter gene-based assays may be conducted in a cell-free manner by contacting a compound with a cell-free extract and a reporter gene construct comprising a reporter gene and a tRNA intron within the open reading frame of the reporter gene or within the 5' untranslated region, 3' untranslated region or both the 5' and 3' untranslated regions of the reporter gene construct, or in a mRNA splicing site of the reporter gene, and measuring the expression of said reporter gene.
  • the alteration in reporter gene expression relative to a previously determined reference range, the absence of the compound or a control in such reporter-gene based assays indicates that a particular compound modulates the activity of an animalia tRNA splicing endonuclease.
  • An increase in reporter gene expression relative to a previously determined reference range, the absence of the compound or a control in such reporter-gene based assays indicates that a particular compound enhances the activity of an animalia tRNA splicing endonuclease.
  • a negative control e.g.
  • a positive control e.g. , an agent that is known to have an effect on the expression of the reporter gene, preferably an agent that effects the activity of an animalia tRNA splicing endonuclease
  • the invention provides a method for identifying a compound that modulates animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting a member of a library of compounds with a cell-free extract and a nucleic acid comprising a reporter gene, wherein the reporter gene comprises a tRNA intron; and (b) detecting the expression of said reporter gene, wherein a compound that modulates tRNA splicing endonuclease activity is identified if the expression of said reporter gene in the presence of a compound is altered relative to the expression of said reporter gene in the absence of said compound or the presence of a control.
  • the activity of a compound in the cell-free extract can be determined by assaying the activity of a reporter protein encoded by a reporter gene, or alternatively, by quantifying the expression of the reporter gene by, for example, labeling the in vitro translated protein (e.g. , with 35 S-labeled methionine), northern blot analysis, RT-PCR or by immunological methods, such as western blot analysis or immunoprecipitation. Such methods are well-known to one of skill in the art.
  • Fluorescence resonance energy transfer can be used to detect alterations in the activity of an animalia tRNA splicing endonuclease.
  • the subunits of an animalia tRNA splicing endonuclease or a substrate for an animalia tRNA splicing endonuclease may be labeled with fluorophores.
  • the substrate for the animalia tRNA splicing endonuclease is labeled with fluorophores.
  • a substrate for an animalia tRNA splicing endonuclease is labeled such that the fluorescent donor moiety and the fluorescent acceptor moiety or a quencher are at most 0.5 nm, at most 1 nm, at most 5 nm, at most 10 nm, at most 20 nm, at most 30 nm, at most 40 nm, at most 50 nm or at most 100 nm apart from each other.
  • the substrates depicted in Figure 1 are used in the FRET assays.
  • the hybridized tRNA substrate and circularly permuted tRNA substrate depicted in Figures 1B and 1C are used in the FRET assays.
  • the free 5' and 3' ends of the intron of the hybridized tRNA substrate ( Figure 1B ) or the free 5' and 3' ends of the intron of circularly permuted tRNA substrate ( Figure 1C ) may be labeled with a fluorophore such that the close spatial proximity of the fluorophore on the 5' end with the fluorophore on the 3' end results in fluorescence resonance energy transfer.
  • Cleavage of the substrate will then result in a spatial separation of the labeled 5' end from the labeled 3' end and thus, in reduced fluorescence resonance energy transfer.
  • the skilled artisan can measure FRET and determine the concentration of cleaved versus uncleaved substrate. The concentration of uncleaved substrate decreases as FRET declines.
  • the 3' end or the 5' end is labeled with a fluorophore and the other end, i.e. , the 5' end or the 3' end, respectively, is labeled with a quencher of the fluorophore.
  • the quencher and the fluorophore are separated from each other resulting in a measurable change in fluorescence.
  • the fluorescence signal increases as the cleavage reaction proceeds.
  • the FRET cell-based assays may be conducted by microinjecting or transfecting (e.g. , using liposomes or electroporation) a substrate for an animalia tRNA splicing endonuclease into a cell and contacting the cell with a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or alternatively, the substrate is labeled at the 5' end with a quencehr and labeled at the 3' end with a fluorophore, and measuring the fluorescence of the substrate by, e.g.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, inhibit or reduce the production of a detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • a compound that enhances the activity of the endogenous endonuclease will enhance the cleavage of the substrate and thus, increase the production of a detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • the FRET cell-based assays maybe conducted by microinj ecting or transfecting a substrate for an animalia tRNA splicing endonuclease into a cell and contacting the cell with a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or, alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g. , fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce cleavage of the substrate and thus, increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g., PBS).
  • a compound that enhances the activity of the endogenous tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g., PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is know to have an effect on the cleavage of the substrate
  • an agent that inhibits or reduces the activity of the animalia tRNA splicing ligase such as an antibody that specifically binds to the ligase may be used in the contacting step to determine, ensure or confirm that a compound is not solely functioning by inhibiting or reducing the activity of the ligase.
  • the FRET cell-based assay may be conducted in cells deficient in tRNA splicing ligase.
  • ATP may be excluded from the assay. Without being bound by theory, since ATP is required for the tRNA splicing ligase reaction, any effect a compound has in the assay should be attributable to an effect of the compound on the endonuclease.
  • the assay can be conducted in any buffer system that provides conditions conducive to the tRNA endonuclease reaction.
  • buffer systems are well known to the skilled artisan.
  • the buffer is the medium in which the cell culture is kept. Care should be taken that Magnesium ions are present in the medium.
  • the assay is conducted for at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) microinjecting or transfecting a substrate of a tRNA splicing endonuclease into a animalia cell, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or alternatively, the substrate is labeled at the 5' end with a quencehr and labeled at the 3' end with a fluorophore; (b) contacting the cell with a member of a library of compounds; and (c) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if a fluorescent signal is not detectable in the presence of the compound relative to the absence
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell containing a substrate of a tRNA splicing endonuclease with a member of a library of compounds, wherein the substrate is labeled at the 5' end with a fluorophore and at the 3' end with a quencher, or alternatively, the substrate is labeled at the 5' end with a quencehr and labeled at the 3' end with a fluorophore; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if a fluorescent signal is not detectable in the presence of the compound relative to the absence of the compound or the presence of a control.
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) microinjecting or transfecting a substrate of a tRNA splicing endonuclease into a animalia cell, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety; (b) contacting the cell with a member of a library of compounds; and (c) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if the fluorescent signal detected in the presence of the compound is altered relative to the absence of the compound
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell containing substrate of a tRNA splicing endonuclease with a member of a library of compounds, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces tRNA splicing activity is identified if the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety in the presence of the compound is reducred relative to the absence of
  • nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease may be utilized as a substrate for an animalia tRNA splicing endonuclease in a FRET assay described herein.
  • a nucleotide sequence comprising a bulge-helix-bulge structure or a mature domain of a precursor tRNA may be utilized as a substrate for an animalia tRNA splicing endonuclease in a FRET assay described herein.
  • a nucleotide sequence recognized and excised by an animalia tRNA splicing endonuclease may comprise 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, or more.
  • the substrates for a tRNA splicing endonuclease utilized in the FRET assays described herein comprise a tRNA intron.
  • the substrate may comprise a bulge-helix-bulge conformation.
  • the substrate comprises a tRNA mature domain that contains an intron.
  • a single pair of fluorescent donor and acceptor moieties can be labeled with different pairs of fluorescent donor moieties and fluorescent acceptor moieties.
  • two, three, four, five or more pairs of fluorescent donor moieties and fluorescent acceptor moieties can be used.
  • at least one of the pairs comprise a fluorescent acceptor moiety that has a different emission spectrum from the fluorescent acceptor moiety of at least one of the other pairs.
  • the fluorescent acceptor moiety of the first pair, second pair and third pair has a different emission spectrum than the fluorescent acceptor moiety of the other two.
  • the labeled substrate can be microinjected or transfected into animalia cells (preferably, mammalian cells and more preferably, human cells) utilizing techniques well-known to one of skill in the art (see, e.g. , Adams et al., 1991, Nature 349:694-697 ).
  • the activity of a compound on an animalia tRNA splicing endonuclease in the FRET cell-based assays can be determined by measuring the fluorescent emission spectra of the substrate utilizing techniques well-known to one of skill in the art. The fluorescent emission spectra measured depends, in part, on the fluorophore used.
  • the FRET cell-free assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease with a cell-free extract (see Section 4.4.1.2 supra regarding cell-free extracts, preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher or, alternatively, the the substrate is labeled at the 3' end with a fluorophore and labeled at the 5' end with a quencher, and measuring the fluorescence of the substrate in, e.g.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease in the cell-free extract will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits or reduces the activity of the tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, inhibit or reduce the production of a detectable fluorescent signal relative to a negative control ( e.g. , PBS).
  • a compound that enhances the activity of the tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, increase the production of a detectable signal relative to a negative control ( e.g. , PBS).
  • the FRET cell-free-based assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease with a cell-free extract (preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that.inhibits or reduces the activity of the tRNA splicing endonuclease will inhibit or reduce cleavage of the substrate and thus, increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS
  • a compound that enhances the activity of the tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • the assay can be conducted in any buffer system that provides conditions conducive to the tRNA endonuclease reaction.
  • buffer systems are well known to the skilled artisan.
  • the buffer comprises 20 mM Tris at a pH of 7.0, 50 mM KC1, 0.1 mM DTT, 5 mM MgCl 2 , and 0.4% Triton X100. Care should be taken that pH, salt concentration, detergent concentration etc. of the buffer system do not interfere with FRET.
  • the assay is conducted for at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell-free extract (preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease with a substrate of a tRNA splicing endonuclease and a member of a library of compounds, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits or reduces t
  • the invention provides a method of identifying an antiproliferative compound that inhibits or reduces animalia tRNA splicing endonuclease activity, said method comprising: (a) contacting an animalia cell-free extract (preferably, a tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease with a substrate of a tRNA splicing endonuclease and a member of a library of compounds, wherein said substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or alternatively, the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety; and (b) measuring the activity of the tRNA splicing endonuclease, wherein an antiproliferative compound that inhibits tRNA
  • the substrate can be labeled with a single pair of fluorescent donor and acceptor moieties.
  • the substrate can be labeled with different pairs of fluorescent donor moieties and fluorescent acceptor moieties.
  • two, three, four, five or more pairs of fluorescent donor moieties and fluorescent acceptor moieties can be used.
  • at least one of.the pairs comprise a fluorescent acceptor moiety that has a different emission spectrum from the fluorescent acceptor moiety of at least one of the other pairs.
  • the fluorescent acceptor moiety of the first pair, second pair and third pair has a different emission spectrum than the fluorescent acceptor moiety of the other two.
  • the activity of a compound on an animalia tRNA splicing endonuclease in the FRET cell-free assays can be determined by measuring the fluorescent emission spectra of the substrate utilizing techniques well-known to one of skill in the art. The fluorescent emission spectra measured depends, in part, on the fluorophore used.
  • a FRET cell-based assay may be conducted by microinjecting or transfecting a first subunit of an animalia tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorophore and a second, different subunit of an animalia tRNA splicing endonuclease ( e.g. , SEN34) labeled with a quencher into a cell and contacting the cell with a compound, and measuring the fluorescence of the animalia tRNA splicing endonuclease by, e.g. , fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the cell microinjected or transfected is deficient in one or more of the subunits of the animalia tRNA splicing endonuclease.
  • Any methods known to the skilled artisan can be used to remove the expression and/or function of one or more subunits of the animalia tRNA splicing endonuclease from the cell.
  • RNAi is used to transiently remove one or more of the subunits of the animalia tRNA splicing endonuclease. The formation of the animalia tRNA splicing endonuclease from the labeled subunits will result in a reduction in the fluorescence detectable.
  • a compound that inhibits or reduces the formation of the animalia tRNA splicing endonuclease will reduce or inhibit the production of a detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • a compound that enhances the formation of the animalia tRNA splicing endonuclease will increase the fluorescence detectable relative to a negative control (e.g. , PBS).
  • a FRET cell-based assay may be conducted by microinjecting a first subunit of an animalia tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorescent donor moiety and a second, different subunit of an animalia tRNA splicing endonuclease ( e.g. , SEN34) labeled with a fluorescent acceptor moiety into a cell and contacting the cell with a compound, and measuring the fluorescence of the animalia tRNA splicing endonuclease by, e.g ., fluorescence microscopy or a fluorescence emission detector such as a Viewlux or Analyst.
  • the formation of the animalia tRNA splicing endonuclease will result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the formation of the animalia tRNA splicing endonuclease will reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control ( e.g. , PBS).
  • a compound that enhances the formation of the animalia tRNA splicing endonuclease will increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • the compound and the cell are incubated for at least 0.2 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or at least 1 day.
  • a FRET cell-free assay may be conducted by contacting a first subunit of an animalia tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorophore and a second subunit of an animalia tRNA splicing endonuclease ( e.g. , SEN34) labeled with a quencher with a compound in vitro under conditions conducive to the formation of the endonuclease, and measuring the fluorescence of the animalia tRNA splicing endonuclease by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the formation of the animalia tRNA splicing endonuclease from the labeled subunits will result in a reduction in the fluorescence detectable.
  • a compound that inhibits or reduces the formation of the animalia tRNA splicing endonuclease will enhance the production of detectable fluorescent signal relative to the absence of the compound or the presence of a negative control ( e.g. , PBS).
  • a compound that enhances the formation of the animalia tRNA splicing endonuclease will reduce or inhibit the fluorescence detectable relative to the absence of the compound or a negative control ( e.g. , PBS).
  • a FRET cell-free assay may be conducted by contacting a first subunit of an animalia tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorescent donor moiety and a second, different subunit of an animalia tRNA splicing endonuclease ( e.g. , SEN34) labeled with a fluorescent acceptor moiety with a compound in vitro under conditions conducive to the formation of the endonuclease, and measuring the fluorescence of the animalia tRNA splicing endonuclease by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the formation of the animalia tRNA splicing endonuclease will result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor.
  • a compound that inhibits or reduces the formation of the animalia tRNA splicing endonuclease will reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to the absence of the compound or the presence of a negative control (e.g. , PBS).
  • a compound that enhances the formation of the animalia tRNA splicing endonuclease will increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to the absence of the compound or the presence of a negative control (e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • Compounds that modulate the activity of an animalia tRNA splicing endonuclease can be identified by direct binding assays.
  • compounds that inhibit the activity of an animalia tRNA splicing endonuclease by directly or indirectly reducing or inhibiting the interaction between a substrate for an animalia tRNA splicing endonuclease and an animalia tRNA splicing endonuclease are described in International Patent Publication Nos. WO 02/083837 and WO 02/083953 , the disclosures of which are hereby incorporated by reference in their entireties.
  • direct binding assays may be conducted by attaching a library of compounds to solid supports, e.g. , polymer beads, with each solid support having substantially one type of compound attached to its surface.
  • the plurality of solid supports of the library is exposed in aqueous solution to a substrate for an animalia tRNA splicing endonuclease having a detectable label, forming a dye-labeled substrate:support-attached compound complex.
  • Binding of a substrate to a particular compound labels the solid support, e.g. , bead, comprising the compound, which can be physically separated from other, unlabeled solid supports.
  • the chemical structures of the compounds thereon can be determined by, e.g. , reading a code on the solid support that correlates with the structure of the attached compound.
  • direct binding assays may be conducted by contacting a substrate for an animalia tRNA splicing endonuclease having a detectable label with a member of a library of compounds free in solution, in labeled tubes or microtiter wells, or a microarray.
  • Compounds in the library that bind to the labeled substrate of an animalia tRNA splicing endonuclease will form a detectably labeled complex that can be identified and removed from the uncomplexed, unlabeled compounds in the library, and from uncomplexed, labeled substrate of an animalia tRNA splicing endonuclease, by a variety of methods including, but not limited to, methods that differentiate changes in the electrophoretic, chromatographic, or thermostable properties of the complexed substrate.
  • the effect of a compound on the activity of an animalia tRNA splicing endonuclease may be determined utilizing a fluorescence polarization-based assay.
  • a fluorescently labeled substrate for an animalia tRNA splicing endonuclease is contacted with an animalia cell-free extract (preferably, an animalia tRNA splicing endonuclease extract) or a purified animalia tRNA splicing endonuclease and a compound or a member of a library of compounds; and the fluorescently polarized light emitted is measured.
  • the size of the substrate used in the assay is large enough to distinguish a change in fluorescent polarized light emitted following cleavage of the substrate.
  • the tRNA splicing endonuclease in the cell-free extract or the purified animalia tRNA splicing endonuclease will cleave the substrate and result in a change in intensity of emitted polarized light.
  • Fluorescently labeled substrates when excited with plane polarized light will emit light in a fixed plane only if they do not rotate during the period between excitation and emission. The extent of depolarization of the emitted light depends upon the amount of rotation of the substrate, which is dependent on the size of the substrate.
  • a small fluorescently labeled substrate rotates rapidly and the emitted light is depolarized.
  • a large fluorescently labeled substrate rotates more slowly and results in the emitted light remaining polarized.
  • a compound that inhibits the activity of the tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, decrease the rotation of the substrate relative to a negative control (e.g ., PBS) or the absence of the compound, which will result in the emitted light remaining polarized.
  • a compound that enhances the activity of the tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, increase the rotation of the substrate relative to a negative control (e.g. , PBS) or the absence of the compound, which will result in more of the emitted light being depolarized.
  • a negative control e.g. , PBS
  • the intensities of the light are measured in planes 90° apart and are many times designated the horizontal and vertical intensities.
  • the excitation filter is moveable while the emission filter is fixed.
  • the horizontal and vertical intensities are measured simultaneously via fiber optics.
  • Fluorescence polarization values are most often divided by 1000 and expressed as millipolarization units (mP).
  • the hybridized tRNA or circularly permuted tRNA depicted in Figure 1 are used as a substrate for the endonuclease.
  • the 5' end in the intron of the hybridized tRNA or the circularly permuted tRNA, or the 3' end in the intron of the hybridized tRNA or the circularly permuted tRNA or both are labeled with a fluorophore.
  • the size of the molecule to which the fluorophore is attached changes because the intron is released from the substrate.
  • the decrease in molecular weight of the labeled molecule results in an increase of depolarization of light that is emitted from the fluorophore. Measuring the amount of depolarization allows the skilled artisan to determine the amount of cleaved substrate.
  • the effect of a compound or a member of a library of compoundson the activity of an animalia tRNA splicing endonuclease may be determined using a tRNA endonuclease suppression assay.
  • a host cell is engineered to contain a first reporter gene construct and a suppressor tRNA; the expression of the suppressor tRNA is induced; the host cell is contacted with a compound or a member of a library of compounds; and the expression of the reporter gene and/or the activity of the protein encoded by the reporter gene is measured.
  • the first reporter gene construct comprises a reporter gene with a nonsense codon in its open reading frame such that the open reading frame is interrupted. Standard mutagenesis techniques described, e.g.
  • the first reporter gene construct is transfected into a host cell engineered to contain a suppressor tRNA.
  • the first reporter gene is cotransfected into a host cell with a suppressor tRNA.
  • the suppressor tRNA's expression is regulated by a controllable regulatory element; such as by a tetracycline regulated regulatory element (see, e.g.
  • the suppressor tRNA contains a tRNA intron in the anticodon stem such that only properly spliced suppressor tRNA is functional.
  • Expression of functional suppressor tRNA is dependent on (i) the transcription of the suppressor tRNA, and (ii) tRNA splicing. The expression of functional suppressor tRNA suppresses the nonsense codon in the reporter gene and results in full length, functional reporter gene expression.
  • full length, functional reporter gene correlates with the expression of functional suppressor tRNA, which in turn correlates with the level of transcription of the suppressor tRNA and tRNA splicing.
  • the expression of full-length reporter gene and the activity of the protein encoded by the reporter gene can be assayed by any method well known to the skilled artisan or as described herein.
  • a compound that inhibits or reduces the activity of an animalia tRNA splicing endonuclease will inhibit or reduce the production of functional suppressor tRNA and thus, reduce the expression of the reporter gene relative to a previously determined reference range or a control.
  • a compound that enhances the activity of an animalia tRNA splicing endonuclease will enhance the production of functional suppressor tRNA and thus, enhance the production of the reporter gene relative to a previously determined reference range or a control.
  • the step of inducing the expression of the suppressor tRNA may be conducted simultaneously with the step of contacting the host cell with a compound or at least 5 minutes, at least 15 minutes, at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours or at least 12 hours before the step of contacting the compound with the host cell.
  • the expression of the suppressor tRNA is induced by incubating the host cell with an agent such as, e.g.
  • tetracycline for approximately 5 minutes, approximately 15 minutes, approximately 0.5 hours, approximately 1 hour, approximately 1.5 hours, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, 6 approximately hours, 8 approximately hours, approximately 10 hours or approximately 12 hours.
  • the host cell is contacted with the compound for approximately 5 minutes, approximately 15 minutes, approximately 0.5 hours, approximately 1 hour, approximately 1.5 hours, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, 6 approximately hours, 8 approximately hours, approximately 10 hours or approximately 12 hours.
  • the host cell is engineered to contain a second reporter gene construct comprising a reporter gene different from the first reporter gene that does not contain a nonsense codon.
  • the reporter genes used in the tRNA endonuclease suppression assay are Red and Green Click Beetle luciferase, wherein the Red luciferase contains the nonsense codon.
  • a host cell may be engineered to stably express the two luciferase genes and the suppressor tRNA whose expression is regulated by a controlled regulatory element (such as a tetracycline controlled regulatory element). In the absence of an agent such as tetracycline, the suppressor tRNA is not expressed and thus the red-to-green ratio is low.
  • the suppressor tRNA In the presence of an agent such as tetracycline, the suppressor tRNA is expressed and thus the red-to-green ratio increases.
  • cells are plated in the presence of a compound. After a certain time-period media containing an agent such as tetracycline is added to induce suppressor tRNA expression.
  • the activity of an animalia tRNA splicing endonuclease may be determined in an assay in which the persistence and quantity of tRNA intron is detected in an animalia cell.
  • the amount of tRNA intron is quantified at different time points after or during the incubation of the cell with the compound.
  • the tRNA intron can be detected by means of Fluorescence in situ hybridization (FISH) using a tRNA intron-specific probe.
  • FISH Fluorescence in situ hybridization
  • a control experiment is conducted in parallel wherein the animalia cell is not contacted with a compound.
  • the splicing reaction In the absence of an inhibitor of animalia tRNA splicing endonuclease, the splicing reaction is fast and the concentration of intron in the cell is low. Without being bound by theory, because the spliced intron is normally degraded the concentration of tRNA intron in the animalia cell is below the detection threshold. In the presence of an inhibitor of animalia tRNA splicing endonuclease, the splicing reaction is slowed down and the amount of tRNA intron increases. Thus, a compound that inhibits animalia tRNA splicing endonuclease can be identified by its ability to increase the level of tRNA intron in the animalia cell.
  • a FISH assay is used to determine the effect of a compound on the activity of an animalia tRNA splicing endonuclease in a high-throughput screen.
  • a 96-lens microscope can be used for a high-throughput screen based on FISH.
  • 96 cell cultures are incubated in a 96-well plate with different compounds. Subsequently, the cells are subjected to a FISH analysis using a tRNA intron specific probe and analyzed using the 96-lens microscope. The presence of a signal or the presence of a significantly stronger signal demonstrates that tRNA intron was present in the cells at elevated levels and thus the compound is a candidate inhibitor of tRNA splicing endonuclease.
  • the FISH assay identifies the compound as inhibitor of the tRNA splicing endonuclease directly.
  • a compound that was identified in a FISH assay as an inhibitor of tRNA splicing is a prima facie candidate for an inhibitor of tRNA splicing endonuclease.
  • the activity of an animalia tRNA splicing endonuclease may be determined in an assay in which the amount of substrate for a tRNA splicing endonuclease cleaved by the endonuclease in the presence of a compound relative to a control (preferably, a negative control and more preferably, a negative control and a positive control) is detected.
  • a control preferably, a negative control and more preferably, a negative control and a positive control
  • Such an assay may be conducted by contacting or incubating a compound with a labeled substrate for an animalia tRNA splicing endonuclease and a cell-free extract or purified animalia tRNA splicing endonuclease under conditions conducive for tRNA splicing endonuclease activity, and measuring the amount of cleaved substrate.
  • the substrate for the animalia tRNA splicing endonuclease can be labeled with any detectable agent.
  • Useful labels in the present invention can include, but are not limited to, spectroscopic labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red, tetramethylrhodimine isothiocynate (TRITC), bora-3a,4a-diaza-s-indacene (BODIPY®) and derivatives, etc .), digoxigenin, biotin, phycoerythrin, AMCA, CyDyeTM, and the like), radiolabels (e.g ., 3 H, 125 I, 35 S, 14 C, 32 P, 33 P, etc .), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc .), spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic (e.g.,
  • a substrate of an animalia tRNA splicing endonuclease can be labeled by any method known to the skilled artisan.
  • a substrate of an animalia tRNA splicing endonuclease can be labeled using site-specific labeling of RNA with fluorophores.
  • a substrate of an animalia tRNA splicing endonuclease is labeled using the methods described in Qin and Pyle, 1999 (Methods 18(1):60-70 ), which is incorporated in its entirety herein.
  • the optimal method for labeling of a substrate of an animalia tRNA splicing endonuclease can be determined by the skilled artisan using routine experimentation.
  • a substrate of an animalia tRNA splicing endonuclease is labeled using different methods, different labels and/or different positions in the tRNA substrate for labeling.
  • the differently labeled substrates are then subjected separately to a splicing assay in the presence and absence, respectively of an inhibitor or an activator of an animalia tRNA splicing endonuclease.
  • the optimal label for the screening assays is the label that allows for the most easily detectable and most reproducible detection of the effect of the inhibitor or the activater.
  • Other labeling procedures may also be used that, for example, provide other desirable advantages.
  • a compound is contacted or incubated with a labeled substrate for an animalia tRNA splicing endonuclease and a cell-free extract or purified animalia tRNA splicing endonuclease for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, or more.
  • the amount of cleaved substrate is proportional to the activity of the tRNA splicing endonuclease.
  • the amount of cleaved tRNA splicing endonuclease can be measured by any technique known to one skilled in the art.
  • the cleaved tRNA splicing endonuclease substrate is separated from the uncleaved tRNA splicing endonuclease substrate by gel-electrophoresis.
  • the amount of cleaved tRNA splicing endonuclease substrate can be quantified by measuring the intensity of the signal of the cleaved tRNA splicing endonuclease substrate. The stronger the signal produced by the cleaved tRNA splicing endonuclease substrate relative to the uncleaved tRNA splicing endonuclease substrate the more active is the tRNA splicing endonuclease.
  • the signal intensity can be quantified using autoradiography or a phosphoimager. If the activity of the tRNA splicing endonuclease is decreased in the presence of a compound, i.e ., if the signal of the cleaved tRNA splicing endonuclease substrate relative to the uncleaved tRNA splicing endonuclease substrate is decreased compared to the reaction without the compound or in the presence of a negative control, the compound is identified as an inhibitor of the tRNA splicing endonuclease.
  • the amount of cleaved tRNA is determined using mass spectrometry.
  • the compound can be deconvoluted, e.g. , by cross-referencing the positive sample to original compound list that was applied to the individual test assays.
  • the sequence of the compound can be determined by direct sequencing of the peptide or nucleic acid. Such methods are well known to one of skill in the art.
  • a number of physico-chemical techniques can be used for the de novo characterization of compounds bound to the target RNA.
  • examples of such techniques include, but are not limited to, mass spectrometry, NMR spectroscopy, X-ray crytallography and vibrational spectroscopy.
  • Mass spectrometry e.g ., electrospray ionization (“ESI”), matrix-assisted laser desorption-ionization (“MALDI”), F and ourier-transform ion cyclotron resonance (“FT-ICR”) can be used for elucidating the structure of a compound.
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption-ionization
  • F ourier-transform ion cyclotron resonance
  • MALDI uses a pulsed laser for desorption of the ions and a time-of-flight analyzer, and has been used for the detection of noncovalent tRNA:amino-acyl-tRNA synthetase complexes ( Gruic-Sovulj et al., 1997, J. Biol. Chem. 272:32084-32091 ).
  • covalent cross-linking between the target nucleic acid and the compound is required for detection, since a non-covalently bound complex may dissociate during the MALDI process.
  • ESI mass spectrometry (“ESI-MS”) has been of greater utility for studying non-covalent molecular interactions because, unlike the MALDI process, ESI-MS generates molecular ions with little to no fragmentation ( Xavier et al., 2000, Trends Biotechnol. 18(8):349-356 ). ESI-MS has been used to study the complexes formed by HIV Tat peptide and protein with the TAR RNA ( Sannes-Lowery et al., 1997, Anal. Chem. 69:5130-5135 ).
  • FT-ICR Fourier-transform ion cyclotron resonance
  • An advantage of mass spectroscopy is not only the elucidation of the structure of the compound, but also the determination of the structure of the compound bound to an RNA. Such information can enable the discovery of a consensus structure of a compound that specifically binds to an RNA.
  • NMR spectroscopy is a valuable technique for identifying complexed target nucleic acids by qualitatively determining changes in chemical shift, specifically from distances measured using relaxation effects, and NMR-based approaches have been used in the identification of small molecule binders of protein drug targets ( Xavier et al., 2000, Trends Biotechnol. 18(8):349-356 ).
  • SAR structure-activity relationships
  • the signal from the bound molecule is monitored by employing line broadening, transferred NOEs and pulsed field gradient diffusion measurements ( Moore, 1999, Curr. Opin. Biotechnol. 10:54-58 ).
  • a strategy for lead generation by NMR using a library of small molecules has been recently described ( Fejzo et al., 1999, Chem. Biol. 6:755-769 ).
  • SAR by NMR can be used to elucidate the structure of a compound.
  • NMR spectroscopy is a technique for identifying binding sites in target nucleic acids by qualitatively determining changes in chemical shift, specifically from distances measured using relaxation effects.
  • Examples of NMR that can be used for the invention include, but are not limited to, one-dimentional NMR, two-dimentional NMR, correlation spectroscopy ("COSY”), and nuclear Overhauser effect (“NOE”) spectroscopy.
  • COSY correlation spectroscopy
  • NOE nuclear Overhauser effect
  • an advantage of NMR is the not only the elucidation of the structure of the compound, but also the determination of the structure of the compound bound to the RNA. Such information can enable the discovery of a consensus structure of a compound that specifically binds to an RNA.
  • X-ray crystallography can be used to elucidate the structure of a compound.
  • the first step in x-ray crystallography is the formation of crystals.
  • the formation of crystals begins with the preparation of highly purified and soluble samples.
  • the conditions for crystallization is then determined by optimizing several solution variables known to induce nucleation, such as pH, ionic strength, temperature, and specific concentrations of organic additives, salts and detergent.
  • Techniques for automating the crystallization process have been developed to automate the production of high-quality protein crystals.
  • crystals are harvested and prepared for data collection.
  • the crystals are then analyzed by diffraction (such as multi-circle diffractometers, high-speed CCD detectors, and detector off-set). Generally, multiple crystals must be screened for structure determinations.
  • Vibrational spectroscopy e.g. infrared (IR) spectroscopy or Raman spectroscopy
  • IR infrared
  • Raman spectroscopy can be used for elucidating the structure of a compound.
  • Infrared spectroscopy measures the frequencies of infrared light (wavelengths from 100 to 10,000 nm) absorbed by the compound as a result of excitation of vibrational modes according to quantum mechanical selection rules which require that absorption of light cause a change in the electric dipole moment of the molecule.
  • the infrared spectrum of any molecule is a unique pattern of absorption wavelengths of varying intensity that can be considered as a molecular fingerprint to identify any compound.
  • Infrared spectra can be measured in a scanning mode by measuring the absorption of individual frequencies of light, produced by a grating which separates frequencies from a mixed-frequency infrared light source, by the compound relative to a standard intensity (double-beam instrument) or pre-measured ('blank') intensity (single-beam instrument).
  • infrared spectra are measured in a pulsed mode ("FT-IR") where a mixed beam, produced by an interferometer, of all infrared light frequencies is passed through or reflected off the compound.
  • FT-IR pulsed mode
  • the resulting interferogram which may or may not be added with the resulting interferograms from subsequent pulses to increase the signal strength while averaging random noise in the electronic signal, is mathematically transformed into a spectrum using Fourier Transform or Fast Fourier Transform algorithms.
  • Raman spectroscopy measures the difference in frequency due to absorption of infrared frequencies of scattered visible or ultraviolet light relative to the incident beam.
  • the incident monochromatic light beam usually a single laser frequency, is not truly absorbed by the compound but interacts with the electric field transiently. Most of the light scattered off the sample will be unchanged (Rayleigh scattering) but a portion of the scatter light will have frequencies that are the sum or difference of the incident and molecular vibrational frequencies.
  • the selection rules for Raman (inelastic) scattering require a change in polarizability of the molecule. While some vibrational transitions are observable in both infrared and Raman spectrometry, must are observable only with one or the other technique.
  • the Raman spectrum of any molecule is a unique pattern of absorption wavelengths of varying intensity that can be considered as a molecular fingerprint to identify any compound.
  • Raman spectra are measured by submitting monochromatic light to the sample, either passed through or preferably reflected off, filtering the Rayleigh scattered light, and detecting the frequency of the Raman scattered light.
  • An improved Raman spectrometer is described in US Patent No. 5,786,893 to Fink et al. , which is hereby incorporated by reference.
  • Vibrational microscopy can be measured in a spatially resolved fashion to address single beads by integration of a visible microscope and spectrometer.
  • a microscopic infrared spectrometer is described in U.S. Patent No. 5,581,085 to Reffner et al. , which is hereby incorporated by reference in its entirety.
  • An instrument that simultaneously performs a microscopic infrared and microscopic Raman analysis on a sample is described in U.S. Patent No. 5,841,139 to Sostek et al. , which is hereby incorporated by reference in its entirety.
  • compounds are synthesized on polystyrene beads doped with chemically modified styrene monomers such that each resulting bead has a characteristic pattern of absorption lines in the vibrational (IR or Raman) spectrum, by methods including but not limited to those described by Fenniri et al., 2000, J. Am. Chem. Soc. 123:8151-8152 .
  • the library of compounds is prepared so that the spectroscopic pattern of the bead identifies one of the components of the compound on the bead. Beads that have been separated according to their ability to bind target RNA can be identified by their vibrational spectrum.
  • appropriate sorting and binning of the beads during synthesis then allows identification of one or more further components of the compound on any one bead.
  • partial identification of the compound on a bead is possible through use of the spectroscopic pattern of the bead with or without the aid of further sorting during synthesis, followed by partial resynthesis of the possible compounds aided by doped beads and appropriate sorting during synthesis.
  • the IR or Raman spectra of compounds are examined while the compound is still on a bead, preferably, or after cleavage from bead, using methods including but not limited to photochemical, acid, or heat treatment.
  • the compound can be identified by comparison of the IR or Raman spectral pattern to spectra previously acquired for each compound in the combinatorial library.
  • the compounds identified in the assays described supra that modulate the activity of an animalia tRNA splicing endonuclease can be further tested for both direct binding to RNA and biological activity.
  • the compounds are tested for biological activity in further assays and/or animal models.
  • the lead compound is used to design congeners or analogs.
  • the lead compound is used to assess the effect on fungal tRNA splicing endonuclease and fungal proliferation.
  • mutagenesis studies can be conducted to assess the mechanism by which a lead compound is modulating the activity of an animalia tRNA splicing endonuclease.
  • the compounds identified in the assays described supra can be tested for biological activity using host cells containing or engineered to contain an animalia tRNA splicing endonuclease coupled to a functional readout system.
  • a phenotypic or physiological readout can be used to assess activity of an animalia tRNA splicing endonuclease in the presence and absence of the lead compound.
  • a phenotypic or physiological readout can be used to assess activity of an animalia tRNA splicing endonuclease in the presence and absence of the lead compound.
  • the animalia tRNA splicing endonuclease may be overexpressed in a cell in which the animalia tRNA splicing endonuclease is endogenously expressed.
  • the effect of a lead compound can be assayed by measuring the cell growth or viability of the target cell.
  • Such assays can be carried out with representative cells of cell types involved in a particular proliferative disorder. A lower level of proliferation or survival of the contacted cells indicates that the lead compound is effective to treat a condition in the patient characterized by uncontrolled cell growth.
  • a lead compound may be screened using cells of a tumor or malignant cell line or an endothelial cell line.
  • cell culture models include, but are not limited to, for lung cancer, primary rat lung tumor cells ( Swafford et al., 1997, Mol. Cell. Biol., 17:1366-1374 ) and large-cell undifferentiated cancer cell lines ( Mabry et al., 1991, Cancer Cells, 3:53-58 ); colorectal cell lines for colon cancer ( Park and Gazdar, 1996, J. Cell Biochem. Suppl. 24:131-141 ); multiple established cell lines for breast cancer ( Hambly et al., 1997, Breast Cancer Res. Treat.
  • cell lines include the cancer cell line Huh7 (human hepatocellular carcinoma cell line) and the cancer cell line Caco-2 (a colon-cancer cell line).
  • the effect of a lead compound on the growth and/or viability of a cancerous cell of a transformed cell is compared to the effect of such a compound on the growth and/or viability of non-cancerous, normal cells.
  • compounds that differentially affect the growth and/or viability of cancerous cells or transformed cells are chosen as anti-proliferative agents.
  • RNA and mRNA and activity can be determined by any method well known in the art.
  • protein can be quantitated by known immunodiagnostic methods such as Western blotting or immunoprecipitation using commercially available antibodies.
  • mRNA can be quantitated using methods that are well known and routine in the art, for example, using northern analysis, RNase protection, the polymerase chain reaction in connection with the reverse transcription.
  • Cell viability can be assessed by using trypan-blue staining or other cell death or viability markers known in the art.
  • the level of cellular ATP is measured to determined cell viability. Differentiation can be assessed, for example, visually based on changes in morphology.
  • the lead compound can also be assessed for its ability to inhibit cell transformation (or progression to malignant phenotype) in vitro.
  • cells with a transformed cell phenotype are contacted with a lead compound, and examined for change in characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo ), for example, but not limited to, colony formation in soft agar, a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, or expression of fetal antigens, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York, pp. 436-446 ).
  • Loss of invasiveness or decreased adhesion can also be assessed to demonstrate the anti-cancer effects of a lead compound.
  • an aspect of the formation of a metastatic cancer is the ability of a precancerous or cancerous cell to detach from primary site of disease and establish a novel colony of growth at a secondary site: The ability of a cell to invade peripheral sites reflects its potential for a cancerous state.
  • Loss of invasiveness can be measured by a variety of techniques known in the art including, for example, induction of E-cadherin-mediated cell-cell adhesion. Such E-cadherin-mediated adhesion can result in phenotypic reversion and loss of invasiveness ( Hordijk et al., 1997, Science 278:1464-66 ).
  • Loss of invasiveness can further be examined by inhibition of cell migration.
  • a variety of 2-dimensional and 3-dimensional cellular matrices are commercially available (Calbiochem-Novabiochem Corp. San Diego, CA). Cell migration across or into a matrix can be examined using microscopy, time-lapsed photography or videography, or by any method in the art allowing measurement of cellular migration.
  • loss of invasiveness is examined by response to hepatocyte growth factor (HGF). HGF-induced cell scattering is correlated with invasiveness of cells such as Madin-Darby canine kidney (MDCK) cells. This assay identifies a cell population that has lost cell scattering activity in response to HGF ( Hordijk et al., 1997, Science 278:1464-66 ).
  • loss of invasiveness can be measured by cell migration through a chemotaxis chamber (Neuroprobe/ Precision Biochemicals Inc. Vancouver, BC).
  • a chemo-attractant agent is incubated on one side of the chamber (e.g. , the bottom chamber) and cells are plated on a filter separating the opposite side (e.g. , the top chamber).
  • the cells In order for cells to pass from the top chamber to the bottom chamber, the cells must actively migrate through small pores in the filter.
  • Checkerboard analysis of the number of cells that have migrated can then be correlated with invasiveness ( see e.g., Ohnishi, T., 1993, Biochem. Biophys. Res. Commun.193:518-25 ).
  • a lead compound is tested for its effects, such as, but not limited to, cytotoxicity, altered gene expression, and altered morphology, on PBMCs (Peripheral Blood Mononuclear Cells).
  • PBMCs Peripheral Blood Mononuclear Cells
  • the lead compounds identified in the assays described herein can be tested for biological activity using animal models for a proliferative disorder. These include animals engineered to contain an animalia tRNA splicing endonuclease coupled to a functional readout system, such as a transgenic mouse. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. In a specific embodiment of the invention, a compound identified in accordance with the methods of the invention is tested in a mouse model system. Such model systems are widely used and well-known to the skilled artisan such as the SCID mouse model or transgenic mice.
  • the anti-angiogenic activity of a compound identified in accordance with the invention can be determined by using various experimental animal models of vascularized tumors.
  • the anti-tumor activity of a compound identified in accordance with the invention can be determined by administering the compound to an animal model and verifying that the compound is effective in reducing the proliferation or spread of cancer cells in said animal model.
  • An example of an animal model for human cancer in general includes, but is not limited to, spontaneously occurring tumors of companion animals (see, e.g. , Vail & MacEwen, 2000, Cancer Invest 18(8):781-92 ).
  • animal models for lung cancer include, but are not limited to, lung cancer animal models described by Zhang & Roth (1994, In Vivo 8(5):755-69 ) and a transgenic mouse model with disrupted p53 function (see, e.g ., Morris et al., 1998, J La State Med Soc 150(4):179-85 ).
  • An example of an animal model for breast cancer includes, but is not limited to, a transgenic mouse that overexpresses cyclin D1 (see, e.g ., Hosokawa et al., 2001, Transgenic Res 10(5):471-8 ).
  • An example of an animal model for colon cancer includes, but is not limited to, a TCRbeta and p53 double knockout mouse (see, e.g. , Kado et al., 2001, Cancer Res 61(6):2395-8 ).
  • animal models for pancreatic cancer include, but are not limited to, a metastatic model of Panc02 murine pancreatic adenocarcinoma (see, e.g. , Wang et al., 2001, Int J Pancreatol 29(1):37-46 ) and nu-nu mice generated in subcutaneous pancreatic tumours (see, e.g. , Ghaneh et al., 2001, Gene Ther 8(3):199-208 ).
  • animal models for non-Hodgkin's lymphoma include, but are not limited to, a severe combined immunodeficiency ("SCID") mouse (see, e.g. , Bryant et al., 2000, Lab Invest 80(4):553-73 ) and an IgHmu-HOX1 1 transgenic mouse (see, e.g. , Hough et al., 1998, Proc Natl Acad Sci USA 95(23):13853-8 ).
  • An example of an animal model for esophageal cancer includes, but is not limited to, a mouse transgenic for the human papillomavirus type 16 E7 oncogene (see, e.g.
  • colorectal carcinomas examples include, but are not limited to, Apc mouse models (see, e.g. , Fodde & Smits, 2001, Trends Mol Med 7(8):369-73 and Kuraguchi et al., 2000, Oncogene 19(50):5755-63 ).
  • the toxicity and/or efficacy of a compound identified in accordance with the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g ., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • Cells and cell lines that can be used to assess the cytotoxicity of a compound identified in accordance with the invention include, but are not limited to, peripheral blood mononuclear cells (PBMCs), Caco-2 cells, and Huh7 cells.
  • PBMCs peripheral blood mononuclear cells
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • a compound identified in accordance with the invention that exhibits large therapeutic indices is preferred. While a compound identified in accordance with the invention that exhibits toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninf
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of a compound identified in accordance with the invention for use in humans.
  • the dosage of such agents lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e ., the concentration of the compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e ., the concentration of the compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the compounds which display the desired biological activity can be used as lead compounds for the development or design of congeners or analogs having useful pharmacological activity.
  • molecular modeling techniques can be used to design variants of the compound that can be more effective.
  • molecular modeling systems are the CHARM and QUANTA programs (Polygen Corporation, Waltham, MA).
  • CHARM performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • Various fungal assays can be conducted to determine the specificity of a lead compound for an animalia tRNA splicing endonuclease. Any of the assays described above with respect to animalia tRNA splicing endonuclease can be used to assess the effect of a lead comound on fungal tRNA splicing endonuclease. Compounds that affect both animalia tRNA splicing endonuclease and fungal tRNA splicing endonuclease may be used to treat, prevent or ameliorate one or more symptoms of cancer and/or a fungal infection in a cancer patient.
  • the FRET cell-based assays may be conducted by microinjecting a substrate for a fungal tRNA splicing endonuclease into a fungal cell and contacting the fungal cell with a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or alternatively the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore, and measuring the fluorescence of the substrate by, e.g ., a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, inhibit or reduce the production of a detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS
  • a compound that enhances the activity of the endogenous endonuclease will enhance the cleavage of the substrate and thus, increase the production of a detectable fluorescent signal relative to a negative control (e.g ., PBS).
  • the FRET cell-based assays may be conducted by microinjecting a substrate for a fungal tRNA splicing endonuclease into a fungal cell and contacting the fungal cell with a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or the substrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the endogenous tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce cleavage of the substrate and thus, increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a compound that enhances the activity of the endogenous tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • nucleotide sequence recognized and excised by a fungal tRNA splicing endonuclease may be utilized as a substrate for a fungal tRNA splicing endonuclease in a FRET assay described herein.
  • a nucleotide sequence comprising a bulge-helix-bulge structure or a mature domain of a precursor tRNA may be utilized as a substrate for a fungal tRNA splicing endonuclease in a FRET assay described herein.
  • a nucleotide sequence recognized and excised by a fungal tRNA splicing endonuclease may comprise 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, or more.
  • the substrates for a tRNA splicing endonuclease utilized in the FRET assays described herein comprise a tRNA intron.
  • the intron may have a bulge-helix-bulge conformation.
  • the nucleotide sequence comprises a mature domain that contains an intron.
  • the substrate can be labeled with a single pair of fluorescent donor and acceptor compounds.
  • the substrate can be labeled with different pairs of fluorescent donor moieties and fluorescent acceptor moieties.
  • two, three, four, five or more pairs of fluorescent donor moieties and fluorescent acceptor moieties can be used.
  • at least one of the pairs comprise a fluorescent acceptor moiety that has a different emission spectrum from the fluorescent acceptor moiety of at least one of the other pairs.
  • the fluorescent acceptor moiety of the first pair, second pair and third pair has a different emission spectrum than the fluorescent acceptor moiety of the other two.
  • the labeled substrate can be microinjected into fungal cells (preferably, yeast) utilized techniques well-known to one of skill in the art.
  • the activity of a compound on a fungal tRNA splicing endonuclease in the FRET cell-based assays can be determined by measuring the fluorescent emission spectra of the substrate utilizing techniques well-known to one of skill in the art. The fluorescent emission spectra measured depends, in part, on the fluorophore used.
  • the FRET cell-free-based assays may be conducted by contacting a substrate for a fungal tRNA splicing endonuclease with a fungal extract (e.g. , a yeast extract) or a purified fungal tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorophore and labeled at the 3' end with a quencher, or alternatively, the substrate is labeled at the 5' end with a quencher and labeled at the 3' end with a fluorophore, and measuring the fluorescence of the substrate by, e.g.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease will cleave the substrate and result in the production of a detectable fluorescent signal.
  • a compound that inhibits or reduces the activity of the tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, inhibit or reduce the production of a detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS
  • a compound that enhances the activity of the endogenous endonuclease will enhance the cleavage of the substrate and thus, increase the production of a detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • the FRET cell-free-based assays may be conducted by contacting a substrate for a fungal tRNA splicing endonuclease with a cell-free extract or a purified fungal tRNA splicing endonuclease and a compound, wherein the substrate is labeled at the 5' end with a fluorescent donor moiety and labeled at the 3' end with a fluorescent acceptor moiety, or the sbustrate is labeled at the 5' end with a fluorescent acceptor moiety and labeled at the 3' end with a fluorescent donor moiety, and measuring the fluorescence of the substrate by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the tRNA splicing endonuclease in the fungal extract will cleave the substrate and result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the activity of the endogenous tRNA splicing endonuclease will inhibit or reduce cleavage of the substrate and thus, increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a compound that enhances the activity of the endogenous tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control (e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • the substate can be labeled with a single pair of fluorescent donor and acceptor moieties.
  • the substrate can be labeled with different pairs of fluorescent donor moieties and fluorescent acceptor moieties.
  • two, three, four, five or more pairs of fluorescent donor moieties and fluorescent acceptor moieties can be used.
  • at least one of the pairs comprise a fluorescent acceptor moiety that has a different emission spectrum from the fluorescent acceptor moiety of at least one of the other pairs.
  • the fluorescent acceptor moiety of the first pair, second pair and third pair has a different emission spectrum than the fluorescent acceptor moiety of the other two.
  • the activity of a compound on a fungal tRNA splicing endonuclease in the FRET fungal extract assays can be determined by measuring the fluorescent emission spectra of the substrate utilizing techniques well-known to one of skill in the art. The fluorescent emission spectra measured depends, in part, on the fluorophore used.
  • an animalia tRNA splicing endonuclease subunit is labeled with a fluorophore and the tRNA substrate is labeled with a fluorophore such that binding of the tRNA substrate to the animalia tRNA splicing endonuclease results in FRET.
  • Compounds can then be assayed for their ability to inhibit FRET. If a compound prevents or reduces FRET between the labeled substrate and the animalia tRNA splicing endonuclease, the compound is identified as an inhibitor of the animalia tRNA splicing endonuclease-tRNA interaction.
  • This compound can then be assayed for its ability to inhibit the endonuclease activity of the animalia tRNA splicing endonuclease by any assay well known to the skilled artisan (see, e.g. , above).
  • a FRET cell-based assay may be conducted by microinjecting or transfecting a first subunit of a fungal tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorophore and a second, different subunit of a fungal tRNA splicing endonuclease ( e.g. , SEN34) labeled with a quencher into a fungal cell and contacting the fungal cell with a compound, and measuring the fluorescence of the fungal tRNA splicing endonuclease by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the cell microinjected or transfected is deficient in one or more of the subunits of the fungal tRNA splicing endonuclease.
  • the formation of the fungal tRNA splicing endonuclease from the labeled subunits will result in a reduction in the fluorescence detectable.
  • a compound that inhibits or reduces the formation of the fungal tRNA splicing endonuclease will enhance the production of detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • a compound that enhances the formation of the fungal tRNA splicing endonuclease will reduce or inhibit the fluorescence detectable relative to a negative control (e.g. , PBS).
  • a FRET cell-based assay may be conducted by microinjecting or transfecting a first subunit of a fungal tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorescent donor moiety and a second, different subunit of a fungal tRNA splicing endonuclease ( e.g. , SEN34) labeled with a fluorescent acceptor moiety into a fungal cell and contacting the fungal cell with a compound, and measuring the fluorescence of the fungal tRNA splicing endonuclease by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the formation of the fungal tRNA splicing endonuclease will result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the formation of the fungal tRNA splicing endonuclease will reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control ( e.g. , PBS).
  • a compound that enhances the formation of the fungal tRNA splicing endonuclease will increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control ( e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • a FRET assay may be conducted by contacting a first subunit of a fungal tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorophore and a second subunit of a fungal tRNA splicing endonuclease ( e.g. , SEN34) labeled with a quencher with a compound in vitro under conditions conducive to the formation of the endonuclease, and measuring the fluorescence of the fungal tRNA splicing endonuclease by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the formation of the fungal tRNA splicing endonuclease from the labeled subunits will result in a reduction in the fluorescence detectable.
  • a compound that inhibits or reduces the formation of the fungal tRNA splicing endonuclease will enhance the production of detectable fluorescent signal relative to a negative control (e.g. , PBS).
  • a compound that enhances the formation of the fungal tRNA splicing endonuclease will reduce or inhibit the fluorescence detectable relative to a negative control (e.g. , PBS).
  • a FRET fungal assay may be conducted by contacting a first subunit of a fungal tRNA splicing endonuclease (e.g. , SEN2) labeled with a fluorescent donor moiety and a second, different subunit of a fungal tRNA splicing endonuclease ( e.g. , SEN34) labeled with a fluorescent acceptor moiety with a compound in vitro under conditions conducive to the formation of the endonuclease, and measuring the fluorescence of the fungal tRNA splicing endonuclease by, e.g. , a fluorescence emission detector such as a Viewlux or Analyst.
  • a fluorescence emission detector such as a Viewlux or Analyst.
  • the formation of the fungal tRNA splicing endonuclease will result in the production of a detectable fluorescent signal by the fluorescent donor moiety and fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety.
  • a compound that inhibits or reduces the formation of the fungal tRNA splicing endonuclease will reduce the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control ( e.g. , PBS).
  • a compound that enhances the formation of the fungal tRNA splicing endonuclease will increase the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to a negative control ( e.g. , PBS).
  • a negative control e.g. , PBS or another agent that is known to have no effect on the cleavage of the substrate
  • a positive control e.g. , an agent that is known to have an effect on the cleavage of the substrate
  • the effect of a compound on the activity of an fungal tRNA splicing endonuclease may be determined utilizing a fluorescence polarization-based assay.
  • a fluorescently labeled substrate for a fungal tRNA splicing endonuclease is contacted with an fungal cell-free extract (preferably, a fungal tRNA splicing endonuclease extract) or a purified fungal tRNA splicing endonuclease and a compound or a member of a library of compounds; and the fluorescently polarized light emitted is measured.
  • the size of the substrate used in the assay is large enough to distinguish a change in fluorescent polarized light emitted following cleavage of the substrate.
  • the fungal tRNA splicing endonuclease in the cell-free extract or the purified fungal tRNA splicing endonuclease will cleave the substrate and result in a change in intensity of emitted polarized light.
  • Fluorescently labeled substrates when excited with plane polarized light will emit light in a fixed plane only if they do not rotate during the period between excitation and emission. The extent of depolarization of the emitted light depends upon the amount of rotation of the substrate, which is dependent on the size of the substrate.
  • Small substrates rotate more than larger substrates between the time they are excited and the time they emit fluorescent light.
  • a small fluorescently labeled substrate rotates rapidly and the emitted light is depolarized.
  • a large fluorescently labeled substrate rotates more slowly and results in the emitted light remaining polarized.
  • a compound that inhibits the activity of the fungal tRNA splicing endonuclease will inhibit or reduce the cleavage of the substrate and thus, decrease the rotation of the substrate relative to a negative control (e.g ., PBS), which will result in the emitted light remaining polarized.
  • a negative control e.g ., PBS
  • a compound that enhances the activity of the fungal tRNA splicing endonuclease will enhance the cleavage of the substrate and thus, increase the rotation of the substrate relative to a negative control (e.g. , PBS), which will result in more of the emitted light being depolarized.
  • a negative control e.g. , PBS
  • the light intensities are measured in planes 90° apart and are many times designated the horizontal and vertical intensities.
  • the excitation filter is moveable while the emission filter is fixed.
  • the horizontal and vertical intensities are measured simultaneously via fiber optics.
  • Research grade fluorescence polarization instruments are commercially available from, e.g. , Pan Vera, BMG Lab Technologies, and LJL Biosystems. Abott provides clinical laboratory instrumentation.
  • polarization intensity vertical - intensity horizontal intensity vertical + intensity horizontal .
  • Fluorescence polarization values are most often divided by 1000 and expressed as millipolarization units (mP).
  • the anti-fungal effect of a lead compound can be assessed utilizing techniques well-known to one of skill in the art.
  • the invention encompasses methods of anti-fungal susceptibility testing as recommended by the National Committee for Clinical Laboratories (NCCLS) (See National Committee for Clinical Laboratories Standards. 1995, Proposed Standard M27T. Villanova, Pa., all of which is incorporated herein by reference in its entirety) and other methods known to those skilled in the art ( Pfaller et al., 1993, Infectious Dis. Clin. N. Am.
  • the invention encompasses determining anti-fungal activities of the lead compounds of the invention using macrodilution methods and/or microdilution methods using protocols known to those skilled in the art (See, Clancy et al., 1997 Journal of Clinical Microbiology, 35(11): 2878-82 ; Ryder et al., 1998, Antimicrobial Agents and Chemotherapy, 42(5): 1057-61 ; U.S. 5,521,153 ; U.S. 5,883,120 , U.S. 5,521,169 , all of which are incorporated by reference in their entirety).
  • a fungal strain is cultured in an appropriate liquid media, and grown at an appropriate temperature, depending on the particular fungal strain used for a determined amount of time, which is also depends on the particular fungal strain used.
  • An innoculum is then prepared photometrically and the turbidity of the suspension is matched to that of a standard, e . g ., a McFarland standard.
  • the effect of the lead compound on the turbidity of the inoculum is determined visually or spectrophotometrically.
  • the minimal inhibitory concentration of the lead compound (MIC) is determined, which is defined as the lowest concentration of the lead compound which prevents visible growth of an inoculum as measured by determining the culture turbidity.
  • the invention also encompasses colorimetric based assays for determining the anti-fungal activity of the lead compounds of the invention.
  • One exemplary colorimetric assay for use in the methods of the invention is described by Pfaller et al. (1994, Journal of Clinical Microbiology, 32(8): 1993-6 , which is incorporated herein by reference in its entirety; also see Tiballi et al., 1995, Journal of Clinical Microbiology, 33(4): 915-7 ).
  • This assay employs a colorimetric endpoint using an oxidation-reduction indicator (Alamar Biosciences, Inc., Sacramento CA).
  • the invention encompasses photometric assays for determining the anti-fungal activity of the lead compounds of the invention using previously described methodology (See Clancy et al., 1997 Journal of Clinical Microbiology, 35(11): 2878-82 ; Jahn et al., 1995, Journal of Clinical Microbiology, 33(3): 661-667 which is incorporated herein by reference in its entirety).
  • This photometric assay is based on quantifying mitochondrial respiration by viable fungi through the reduction of 3-(4,5-dimethyl-2thiazolyl)-2,5,-diphenyl-2H-tetrazolium bromide (MTT) to formazan.
  • MIC's determined by this assay are defined as the highest concentration of the lead compound associated with the first precipitous drop in optical density.
  • the compounds of the invention are assayed for anti-fungal activity using macrodilution, microdilution and MTT assays in parallel.
  • the antifungal properties of the lead compounds of the present invention may be determined from a fungal lysis assay, as well as by other methods, including, inter alia , growth inhibition assays, fluorescence-based fungal viability assays, flow cytometry analyses, and other standard assays known to those skilled in the art.
  • the fungi tested in accordance with the invention include, but are not limited to fungi in the genus Blastomyces , including Blastomyces dermatitidis ; Paracoccidiodes , including Paracoccidioides brasiliensis ; Sporothrix , including Sporothrix schenckii ; Cryptococcus ; Candida , including Candida albicans , Candida tropicalis and Candida glabrala ; Aspergillus , including Aspergillus fumigarus and Aspergillus flavus ; Histoplasma , including Histoplasma capsulatum ; Cryptococcus , including Cryptococcus neoformans ; Bipolaris ; Cladophialophora ; Cladosporium ; Drechslera ; Exophiala; Fonsecaea ; Phialophora ; Xylohypha ; Ochroconis ; Rhinocladiella ; Scolecobas
  • the subunit(s) of an animalia tRNA splicing endonuclease and/or the nucleotide sequence of a substrate for an animalia tRNA splicing endonuclease that are necessary for a compound identified in accordance with the methods of the invention to modulate the activity of an animalia tRNA splicing endonuclease can be determined utilizing standard mutagenesis techniques well-known to one of skill in the art.
  • One or more mutations may be introduced into an animalia tRNA splicing endonuclease subunit and the effect of the mutations on the activity of the animalia tRNA splicing endonuclease in the presence or absence of a compound can be determined using an assay described herein.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence of an animalia tRNA splicing endonuclease and/or the nucleotide sequence of a substrate for an animalia tRNA splicing endonuclease, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis.
  • less than 75 nucleic acid residue substitutions are introduced into the nucleotide sequence of an animalia tRNA splicing endonuclease and/or the nucleotide sequence of a substrate for an animalia tRNA splicing endonuclease.
  • the present invention provides methods of preventing, treating, managing or ameliorating a proliferative disorder or one or more symptoms thereof, said methods comprising administering to a subject in need thereof one or more compounds identified in accordance with the methods of the invention.
  • the invention provides a method of preventing, treating, managing or ameliorating a proliferative disorder or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of one or more compounds identified in accordance with the methods of the invention.
  • a compound identified in accordance with the methods of the invention is not administered to prevent, treat, or ameliorate a proliferative disorder or one or more symptoms thereof, if such compound has been used previously to prevent, treat, manage or ameliorate said proliferative disorder.
  • the invention also provides methods of preventing, treating, managing or ameliorating a proliferative disorder or one or more symptoms thereof, said methods comprising administering to a subject in need thereof one or more of the compounds identified utilizing the screening methods described herein, and one or more other therapies (e.g ., prophylactic or therapeutic agents), which therapies are currently being used, have been used or are known to be useful in the prevention, treatment, management or amelioration of one or more symptoms associated with said proliferative disorder (including, but not limited to the prophylactic or therapeutic agents listed in Section 4.6.1 hereinbelow).
  • the therapies (e . g ., prophylactic or therapeutic agents) of the combination therapies of the invention can be administered sequentially or concurrently.
  • the combination therapies of the invention comprise a compound identified in accordance with the invention and at least one other therapy that has the same mechanism of action as said compound.
  • the combination therapies of the invention comprise a compound identified in accordance with the methods of the invention and at least one other therapy (e.g ., prophylactic or therapeutic agent) which has a different mechanism of action than said compound.
  • the combination therapies of the present invention improve the prophylactic or therapeutic effect of a compound of the invention by functioning together with the compound to have an additive or synergistic effect.
  • the combination therapies of the present invention reduce the side effects associated with the therapies ( e . g ., prophylactic or therapeutic agents).
  • the prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition.
  • the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions.
  • the prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.
  • a pharmaceutical composition comprising one or more compounds identified in a screening assay described herein is administered to a subject, preferably a human, to prevent, treat, manage or ameliorate a proliferative disorder or one or more symptoms thereof.
  • the pharmaceutical composition may also comprise one or more prophylactic or therapeutic agents which are currently being used, have been used or are known to be useful in the prevention, treatment, management or amelioration of a proliferative disorder or one or more symptoms thereof.
  • a compound identified in accordance with the methods of the invention may be used as a first, second, third, fourth or fifth line of therapy for a proliferative disorder.
  • the invention provides methods for treating, managing or ameliorating a proliferative disorder or one or more symptoms thereof in a subject refractory to conventional therapies for such proliferative disorder, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a compound identified in accordance with the methods of the invention.
  • a cancer may be determined to be refractory to a therapy means when at least some significant portion of the cancer cells are not killed or their cell division arrested in response to the therapy.
  • a determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context.
  • a cancer is refractory where the number of cancer cells has not been significantly reduced, or has increased.
  • the invention provides methods for treating, managing or ameliorating one or more symptoms of a proliferative disorder in a subject refractory to existing single agent therapies for such proliferative disorder, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a compound identified in accordance with the methods of the invention and a dose of a prophylactically or therapeutically effective amount of one or more other therapies ( e.g ., prophylactic or therapeutic agents).
  • the invention also provides methods for treating or managing a proliferative disorder by administering a compound identified in accordance with the methods of the invention in combination with any other therapy ( e .
  • the invention also provides methods for the treatment or management of a patient having a proliferative disorder and immunosuppressed by reason of having previously undergone other therapies.
  • the invention also provides alternative methods for the treatment or management of a proliferative disorder such as cancer where chemotherapy, radiation therapy, hormonal therapy, and/or biological therapy/immunotherapy has proven or may prove too toxic, i . e. , results in unacceptable or unbearable side effects, for the subject being treated or managed.
  • the invention provides methods for preventing the recurrence of a proliferative disorder such as cancer in patients that have been treated and have no disease activity by administering a compound identified in accordance with the methods of the invention.
  • Proliferative disorders that can be treated by the methods encompassed by the invention include, but are not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth (e.g ., psoriasis and pulmonary fibrosis).
  • the cancer may be a primary or metastatic cancer.
  • Specific examples of cancers that can be treated by the methods encompassed by the invention include, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain.
  • Additional cancers include, but are not limited to, the following: leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Walden
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B.
  • cancers caused by aberrations in apoptosis can also be treated by the methods and compositions of the invention.
  • Such cancers may include, but not be limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.
  • the present invention provides methods of preventing, treating, managing or ameliorating cancer or one or more symptoms thereof, said methods comprising administering to a subject in need thereof one or more compounds identified in accordance with the methods of the invention and one or more therapies (e . g ., prophylactic or therapeutic agents).
  • Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • Any therapy e.g ., chemotherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies
  • chemotherapies e.g., radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies
  • agents i.e., anti-cancer agents
  • agents include, but are not limited to, angiogenesis inhibitors, topoisomerase inhibitors and immunomodulatory agents (such as chemotherapeutic agents).
  • Angiogenesis inhibitors include, but are not limited to, angiostatin (plasminogen fragment); antiangiogenic antithrombin III; angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; combretastatin A-4; endostatin (collagen XVIII fragment); fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinase inhibitors (TIMPs); 2-methoxyestradi
  • anti-cancer agents which can be used in accordance with the methods of the invention include, but not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin
  • anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
  • the invention also encompasses the administration of one or more compounds identified in accordance with the methods of the invention in combination with radiation therapy comprising the use of x-rays, gamma rays and other sources of radiation to destroy the cancer cells.
  • the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment is administered as internal therapy or brachytherapy wherein a radiaoactive source is placed inside the body close to cancer cells or a tumor mass.
  • Biologically active compounds identified using the methods of the invention or a pharmaceutically acceptable salt thereof can be administered to a patient, preferably a mammal, more preferably a human, suffering from a proliferative disorder.
  • a compound or a pharmaceutically acceptable salt thereof is administered to a patient, preferably a mammal, more preferably a human, as a preventative measure against a proliferative disorder.
  • the compound or a pharmaceutically acceptable salt thereof is preferably administered as component of a composition that optionally comprises a pharmaceutically acceptable vehicle.
  • the composition can be administered orally, or by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings ( e.g ., oral mucosa, rectal, and intestinal mucosa, etc .) and may be administered together with another biologically active agent. Administration can be systemic or local.
  • Various delivery systems are known, e . g ., encapsulation in liposomes, microparticles, microcapsules, capsules, etc ., and can be used to administer the compound and pharmaceutically acceptable salts thereof.
  • Methods of administration include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the mode of administration is left to the discretion of the practitioner. In most instances, administration will result in the release of the compound or a pharmaceutically acceptable salt thereof into the bloodstream.
  • This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g ., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g ., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.
  • the compound and pharmaceutically acceptable salts thereof can be formulated as a suppository, with traditional binders and vehicles such as triglycerides.
  • the compound and pharmaceutically acceptable salts thereof can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533 ; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989 ); Lopez-Berestein, ibid., pp. 317-327 ; see generally ibid.).
  • a liposome see Langer, 1990, Science 249:1527-1533 ; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989 ); Lopez-Berestein, ibid., pp. 317-327 ; see generally ibid.).
  • the compound and pharmaceutically acceptable salts thereof can be delivered in a controlled release system (see, e.g ., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984 )).
  • a controlled release system discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.
  • a pump may be used (see Langer, supra ; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al., 1980, Surgery 88:507 ; Saudek et al., 1989, N. Engl. J. Med. 321:574 ).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974 ); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984 ); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 ; see also Levy et al., 1985, Science 228:190 ; During et al., 1989, Ann. Neurol. 25:351 ; Howard et al., 1989, J. Neurosurg. 71:105 ).
  • a controlled-release system can be placed in proximity of a target RNA of the compound or a pharmaceutically acceptable salt thereof, thus requiring only a fraction of the systemic dose.
  • compositions comprising the compound or a pharmaceutically acceptable salt thereof (“compound compositions”) can additionally comprise a suitable amount of a pharmaceutically acceptable vehicle so as to provide the form for proper administration to the patient.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, mammals, and more particularly in humans.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered.
  • Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • the pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compound of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.
  • Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Compound compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compound compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the pharmaceutically acceptable vehicle is a capsule (see e.g ., U.S. Patent No. 5,698,155 ).
  • suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro, ed., Mack Publishing Co. Easton, PA, 19th ed., 1995, pp. 1447 to 1676 , incorporated herein by reference.
  • compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions may contain one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions.
  • fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
  • delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
  • a time delay material such as glycerol monostearate or glycerol stearate may also be used.
  • compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such vehicles are preferably of pharmaceutical grade.
  • compositions for intravenous administration comprise sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent.
  • the compound or a pharmaceutically acceptable salt thereof can be formulated for intravenous administration.
  • Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to lessen pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the compound or a pharmaceutically acceptable salt thereof is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the amount of a compound or a pharmaceutically acceptable salt thereof that will be effective in the treatment of a particular disease will depend on the nature of the disease, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for oral administration are generally about 0.001 milligram to about 500 milligrams of a compound or a pharmaceutically acceptable salt thereof per kilogram body weight per day.
  • the oral dose is about 0.01 milligram to about 100 milligrams per kilogram body weight per day, more preferably about 0.1 milligram to about 75 milligrams per kilogram body weight per day, more preferably about 0.5 milligram to 5 milligrams per kilogram body weight per day.
  • the dosage amounts described herein refer to total amounts administered; that is, if more than one compound is administered, or if a compound is administered with a therapeutic agent, then the preferred dosages correspond to the total amount administered.
  • Oral compositions preferably contain about 10% to about 95% active ingredient by weight.
  • Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 milligram to about 100 milligrams per kilogram body weight per day, about 0.1 milligram to about 35 milligrams per kilogram body weight per day, and about 1 milligram to about 10 milligrams per kilogram body weight per day.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight per day to about 1 mg/kg body weight per day.
  • Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight per day and comprise active ingredient in the range of about 0.5% to about 10% by weight.
  • Suitable dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation are in the range of about 0.001 milligram to about 200 milligrams per kilogram of body weight per day.
  • Suitable doses for topical administration are in the range of about 0.001 milligram to about 1 milligram, depending on the area of administration.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.
  • the compound and pharmaceutically acceptable salts thereof are preferably assayed in vitro and in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays can be used to determine whether it is preferable to administer the compound, a pharmaceutically acceptable salt thereof, and/or another therapeutic agent.
  • Animal model systems can be used to demonstrate safety and efficacy.

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